CN112513071A - Gene therapy constructs and methods of use - Google Patents
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- CN112513071A CN112513071A CN201980029113.5A CN201980029113A CN112513071A CN 112513071 A CN112513071 A CN 112513071A CN 201980029113 A CN201980029113 A CN 201980029113A CN 112513071 A CN112513071 A CN 112513071A
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Abstract
Provided herein are improved gene therapy vectors and methods of use, which in some embodiments comprise sequences for improving expression and cell targeting of therapeutic proteins.
Description
Cross referencing
The present application claims the benefit of U.S. provisional application No. 62/664,741 filed on 30.4.2018, U.S. provisional application No. 62/688,640 filed on 22.6.2018, and U.S. provisional application No. 62/744,068 filed on 10.10.2018, each of which is incorporated herein by reference in its entirety.
Background
Genetic disorders occur through heritable or nascent mutations that occur in the coding regions of genes of the genome. In some cases, such genetic disorders are treated by administering a protein encoded by a gene that is mutated in an individual having the genetic disorder. However, such treatment is challenging as administration of the protein does not always result in the protein reaching the organ, cell or organelle in which it is needed. In addition, such treatments typically require infusion once every two weeks, whereas gene therapy does not, where a single treatment can provide long-lasting relief. Thus, gene therapy is likely to provide improved results compared to currently available treatments for genetic disorders.
Disclosure of Invention
Provided herein are compositions and methods for treating genetic disorders using gene therapy. Also provided herein are gene therapy vector compositions and methods for gene therapy for improving protein expression and increasing cellular uptake or delivery and intracellular or subcellular targeting of therapeutic proteins provided by gene therapy vectors.
In certain aspects, gene therapy vectors are provided, e.g., gene therapy vectors comprising a nucleic acid construct, the nucleusThe acid construct comprises, in 5 'to 3' order: (a) a translation initiation sequence, and (b) a nucleic acid sequence encoding a therapeutic protein. In some embodiments, the translation initiation sequence comprises a Kozak sequence. In some embodiments, the translation initiation sequence and the nucleic acid sequence encoding the therapeutic protein may overlap such that the last three nucleotides of the translation initiation sequence are also the start codon of the therapeutic protein. In some embodiments, the Kozak sequence comprises the sequence AX1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of which is any nucleotide. In some embodiments, X1Contains A. In some embodiments, X2Contains G. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence differs from the sequence of AAGATGA (SEQ ID NO:29) by one or two nucleotides. In some embodiments, the Kozak sequence comprises AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence comprises a nucleic acid sequence at least 85% identical to GCAAGATG (SEQ ID NO:44), wherein the last three nucleotides (ATG) are also the start codon of the therapeutic protein. In some embodiments, the Kozak sequence differs from the sequence of GCAAGATG (SEQ ID NO:44) by one or two nucleotides. In some embodiments, the Kozak sequence comprises GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to CACCATG (SEQ ID NO: 47). In some embodiments, the Kozak sequence differs from the sequence of CACCATG (SEQ ID NO:47) by one or two nucleotides. In some embodiments, the Kozak sequence comprises CACCATG (SEQ ID NO: 47). In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a signal peptide capable of increasing secretion of the therapeutic protein compared to the therapeutic protein without the signal peptide. In some embodiments, the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide. In some embodiments, the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17. In some embodiments, the signal peptide is identical to a sequence selected from the group consisting of SEQ ID Nos. 13-17 By 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid. In some embodiments, the BiP signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 13-17. In some embodiments, the Gaussia signal peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32. In some embodiments, the signal peptide differs from the sequence of SEQ ID No. 32 by 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid. In some embodiments, the Gaussia signal peptide comprises the amino acid sequence of SEQ ID NO 32. In some embodiments, the nucleic acid construct further comprises an Internal Ribosome Entry Sequence (IRES). In some embodiments, the IRES is a cricket paralysis virus (CrPV) IRES. In some embodiments, the IRES comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO 12. In some embodiments, the IRES comprises SEQ ID NO 12.
In a further aspect, there is provided a gene therapy vector comprising a nucleic acid construct comprising, in 5 'to 3' order: (a) a nucleic acid sequence encoding a signal peptide, and (b) a nucleic acid sequence encoding a therapeutic protein, wherein the signal peptide is capable of increasing secretion of the therapeutic protein compared to the therapeutic protein without the signal peptide. In some embodiments, the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide. In some embodiments, the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17. In some embodiments, the signal peptide differs from a sequence selected from the group consisting of SEQ ID Nos. 13-17 by 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid. In some embodiments, the BiP signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 13-17. In some embodiments, the signal peptide comprises a Gaussia signal peptide. In some embodiments, the Gaussia signal peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32. In some embodiments, the signal peptide differs from the sequence of SEQ ID No. 32 by 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid. In some embodiments In one embodiment, the Gaussia signal peptide comprises SEQ ID NO 32. In some embodiments, the nucleic acid construct further comprises a translation initiation sequence. In some embodiments, the translation initiation sequence comprises a Kozak sequence comprising AX1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of which is any nucleotide. In some embodiments, X1Contains A. In some embodiments, X2Contains G. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence differs from the sequence of AAGATGA (SEQ ID NO:29) by one or two nucleotides. In some embodiments, the Kozak sequence comprises AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence differs from the sequence of GCAAGATG (SEQ ID NO:44) by one or two nucleotides. In some embodiments, the Kozak sequence comprises GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to CACCATG (SEQ ID NO: 47). In some embodiments, the Kozak sequence differs from the sequence of CACCATG (SEQ ID NO:47) by one or two nucleotides. In some embodiments, the Kozak sequence comprises CACCATG (SEQ ID NO: 47). In some embodiments, the nucleic acid construct further comprises an Internal Ribosome Entry Sequence (IRES). In some embodiments, the IRES comprises an IRES selected from the group consisting of: cricket paralysis virus (CrPV) IRES, picornavirus IRES, foot and mouth disease virus IRES, Kaposi's sarcoma-associated herpesvirus IRES, hepatitis a IRES, hepatitis c IRES, pestivirus IRES, cricket paralysis virus (criptavirus) IRES, Rhopalosiphum palustris (Rhopalosiphum padi) virus IRES, and Merek's disease virus IRES. In some embodiments, the IRES comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO 12. In some embodiments, the IRES comprises SEQ ID NO 12.
In other aspects, gene therapy vectors are provided comprising a nucleic acid construct comprising, in 5 'to 3' orderComprises the following components: (a) an Internal Ribosome Entry Sequence (IRES), and (b) a nucleic acid sequence encoding a therapeutic protein. In some embodiments, the IRES comprises an IRES selected from the group consisting of: cricket paralysis virus (CrPV) IRES, picornavirus IRES, foot and mouth disease virus IRES, Kaposi sarcoma-associated herpesvirus IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, cricket paralysis virus IRES, gloomycosis graminearum virus IRES, and Merrex virus IRES. In some embodiments, the IRES is a cricket paralysis virus (CrPV) IRES. In some embodiments, the IRES comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO 12. In some embodiments, the IRES comprises SEQ ID NO 12. In some embodiments, the nucleic acid construct further comprises a translation initiation sequence. In some embodiments, the translation initiation sequence comprises a Kozak sequence comprising AX1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of which is any nucleotide. In some embodiments, X1Contains A. In some embodiments, X 2Contains G. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence comprises AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence differs from the sequence of GCAAGATG (SEQ ID NO:44) by one or two nucleotides. In some embodiments, the Kozak sequence comprises GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to CACCATG (SEQ ID NO: 47). In some embodiments, the Kozak sequence differs from the sequence of CACCATG (SEQ ID NO:47) by one or two nucleotides. In some embodiments, the Kozak sequence comprises CACCATG (SEQ ID NO: 47). In some embodiments, the nucleic acid construct further comprises a signal nucleic acid sequence encoding a signal peptide capable of increasing secretion of the therapeutic protein compared to the therapeutic protein without the signal peptide. In some embodiments, the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide. In some cases In embodiments, the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17. In some embodiments, the BiP signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 13-17. In some embodiments, the Gaussia signal peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32. In some embodiments, the Gaussia signal peptide comprises SEQ ID NO 32.
In some embodiments, any of the nucleic acid constructs provided herein further comprises a nucleic acid sequence encoding a peptide that selectively binds CI-MPR with high affinity, wherein the therapeutic protein and the peptide that selectively binds CI-MPR are expressed as a fusion protein. In some embodiments, the nucleic acid construct further comprises a sequence encoding a linker peptide between the nucleic acid encoding the peptide that selectively binds to the CI-MPR nucleotide sequence and the nucleic acid sequence encoding the therapeutic protein. In some embodiments, the sequence of the linker peptide may overlap with the sequence of the therapeutic peptide or the sequence of the peptide that selectively binds to CI-MPR, or both. In some embodiments, the peptide that binds with high affinity to CI-MPR is a variant IGF2 peptide (vIGF 2). In some embodiments, the vIGF2 peptide facilitates uptake into cells. In some embodiments, the vIGF2 peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 1-11. In some embodiments, the vIGF2 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 2-11. In some embodiments, the vIGF2 nucleotide sequence is 5' to the nucleic acid sequence encoding the therapeutic protein. In some embodiments, the vIGF2 nucleotide sequence is 3' to the nucleic acid sequence encoding the therapeutic protein. In some embodiments, the nucleic acid construct further comprises a sequence encoding a linker peptide between said vIGF2 nucleotide sequence and said nucleic acid sequence encoding a therapeutic protein. In some embodiments, the linker peptide consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO 18-21, SEQ ID NO 33, or SEQ ID NO 37. In some embodiments, the linker peptide comprises SEQ ID NO 18-21, SEQ ID NO 33, or SEQ ID NO 37. In some embodiments, the therapeutic protein is associated with a lysosomal storage disease. In some embodiments, the therapeutic protein is a lysosomal enzyme or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is selected from the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein is an alpha-galactosidase, or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is an alpha-glucosidase, or an enzymatically active fragment thereof. In some embodiments, the therapeutic protein is a Palmitoyl Protein Thioesterase (PPT), including palmitoyl protein thioesterases 1 and 2 (PPT 1 and PPT2, respectively). In some embodiments, the therapeutic protein is palmitoyl protein thioesterase 1. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the therapeutic protein is associated with a genetic disorder selected from the group consisting of: CDKL5 deficiency, cystic fibrosis, alpha-thalassemia and beta-thalassemia, sickle cell anemia, Marfan syndrome, fragile X syndrome, Huntington's disease, hemochromatosis, congenital deafness (non-syndromic), Tay-saxose (Tay-Sachs), familial hypercholesterolemia, Duchenne muscular dystrophy (Duchenne muscular dystrophy), Stargardt disease (Stargardt disease), User syndrome, choroideremia, achromatopsia, X-linked retinoschisis, hemophilia, Weather-Austenitis syndrome (Wiskott-Aldrich syndrome), X-linked chronic granulomatosis, aromatic L-amino acid decarboxylase deficiency, recessive dystrophic epidermolysis bullosa, alpha 1 antitrypsin deficiency, Husky-early syndrome (HG-early-wish syndrome), Gilson disease, Gilson-Harq syndrome, Gilson-Hardy's disease, Gilson-Daphse syndrome, Gilson syndrome, Sjohnson-induced deficiency, Sjohnson syndrome, S, Noonan syndrome (Noonan syndrome), severe combined immunodeficiency X-SCID. In some embodiments, the therapeutic protein is selected from the group consisting of: CDKL5, connexin 26, hexosidase A, LDL receptor, dystrophin, CFTR, β -globin, HFE, huntingtin, ABCA4, myosin VIIA (MYO7A), Rab convoyin-1 (REP1), cyclic nucleotide-gated channel β 3(CNGB3), retinoschisin 1(RS1), heme subunit β (HBB), factor IX, WAS, cytochrome B-245 β chain, Dopa Decarboxylase (DDC), collagen type VII α 1 chain (COL7a1), serine protease inhibitor family a member 1(SERPINA1), LMNA, PTPN11, SOS1, RAF1, KRAS, and IL2 receptor γ genes. In some embodiments, the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucosaminuria, Barretch disease, cystinosis, Fabry disease (Fabry disease), Gaucher disease type I (Gaucher disease), Gaucher disease type II, Gaucher disease type III, Pompe disease (Pompe disease), Tay Sachs disease (Tay Sachs disease), Mulberry Hoff disease (Sandhoff disease), metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Herle disease (Hurler disease), Hunter disease (Hunter disease), Sannippo disease type A (Sannnippo disease), Sannippo disease type B, Sannippy disease type C, Murphy disease type D, Morquio disease type A (Morquio disease), Moraxe disease type B, Quassie disease (Mardie disease), and Mentius-Pimpire disease (horse-Pick disease), Nippon disease type A (Pimpinesis disease), Moraxe disease type A (horse disease-Pick disease), Moraxe disease type A-Nipple disease (horse disease-Pick disease), Morchelle disease type A, Sandhoff disease, Sandperce disease, Murphy disease, and Morse type D, Niemann-pick disease type C1, niemann-pick disease type C2, sinderler disease type I (Schindler disease), and sinderler disease type II. In some embodiments, the lysosomal storage disease is selected from the group consisting of: deficiency of activating factor, GM 2-gangliosidosis; GM 2-gangliosidosis, AB variant; alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe); beta-mannoside storage disease; lysosomal acid lipase deficiency; cystinosis (type of late onset juvenile or juvenile nephropathy; nephropathic nature in infants); Chanarin-Dorfman syndrome (Chanarin-Dorfman syndrome); neutral lipid storage disease with myopathy; NLSDM; danong disease (Danon disease); fabry disease; late onset Fabry disease type II; faber disease (Farber disease); farber fatty granulomatosis; fucoside storage disorders; galactosialistorage disorder (combined neuraminidase and beta-galactosidase deficiency); gaucher disease; gaucher disease type II; gaucher disease type III; gaucher disease type IIIC; atypical gaucher disease due to sphingolipid activator protein C deficiency; GM 1-gangliosidosis (late-onset/juvenile GM 1-gangliosidosis; adult/chronic GM 1-gangliosidosis); globular cell leukodystrophy, Krabbe disease (late onset infant; juvenile onset; adult onset); atypical krabbe's disease due to sphingolipid activator protein a deficiency; metachromatic leukodystrophy (juvenile; adult); partial sulfaterebroside deficiency; pseudoarylsulfatase a deficiency; metachromatic leukodystrophy due to sphingolipid activator B deficiency; mucopolysaccharide storage disorder: MPS I, heler syndrome; MPS I, heler-schey syndrome; MPS I, schey syndrome; MPS II, hunter syndrome; MPS II, hunter syndrome; sanfilippo syndrome/MPS IIIA type a; sanfilippo syndrome/MPS IIIB type B; sanfilippo syndrome/MPS IIIC type C; sanfilippo syndrome/MPS IIID type D; morquio syndrome, type a/MPS IVA; morquio syndrome, type B/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI mallotte-Lamy syndrome (Maroteaux-Lamy syndrome); MPS VII stri syndrome (Sly syndrome); salivary gland disease type I, II mucolipidosis; i-cell disease, Leroy disease (Leroy disease), mucolipidosis II; shampooing malnutrition/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA; type IV mucolipidosis; polythiol esterase deficiency; niemann-pick disease (type B; type C1/chronic neurological form; type C2; type D/Nova scotia); neuronal ceroid lipofuscinosis: CLN6 disease, atypical late infant, late onset variant, early childhood; Batten-Spielmeyer-Vogt/juvenile NCL/CLN3 disease; finnish variant late infant CLN 5; Jansky-Bielschowsky disease/late infant CLN2/TPP 1; kufs (Kufs)/NCL adult onset/CLN 4 disease (type B); northern epilepsy/variant late infant CLN 8; Santavario-Haldiya (Santavuori-Haltia)/infantile CLN1/PPT disease; pompe disease (glycogen storage disease type II); late onset pompe disease; dense bone developmental disorder; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sinderler disease (type III/moderate, variable); shenqi disease (Kanzaki disease); sala disease (sala disease); infantile asialostorage disease (ISSD); spinal muscular atrophy with progressive myospasm epilepsy (SMAPME); Tay-saxophone/GM 2 gangliosidosis; juvenile onset tay-saxophone disease; late onset tay-saxophone; christian syndrome (Christianson syndrome); laowei (Lowe) eye-brain-kidney syndrome; 4J type Charcot-Marie-dus disease (Charcot-Marie-Tooth), CMT 4J; the Yuris-Varon syndrome (Yunis-Varon syndrome); bilateral temporal occipital polycephalic gyra (BTOP); x-linked hypercalcemic nephrolithiasis, dent-1; and dengue disease 2, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID) and neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is pompe disease. In some embodiments, the genetic disorder is neuronal ceroid lipofuscinosis. In some embodiments, the neuronal ceroid lipofuscinosis is selected from the group consisting of: infant NCL (Santaovri-Hardy's disease), advanced infant NCL (Janski-Bilski disease), Barteng disease, adult NCL (Kofski disease), Finland advanced infant NCL, variant advanced infant NCL, CLN7, CLN8, Turkey advanced infant NCL, NCL 9, and CLN 10. In some embodiments, the gene therapy vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a poxviral vector, a vaccinia viral vector, an adenoviral vector, or a herpesvirus vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector comprises an Inverted Terminal Repeat (ITR). In some embodiments, the AAV vector is selected from the group consisting of: AAV1 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector, AAV8 vector, AAV9 vector, AAVrhS vector, AAVrh10 vector, AAVrh33 vector, AAVrh34 vector, AAVrh74 vector, AAV Anc80 vector, aavphp.b vector, AAVhu68 vector and AAV-DJ vector.
In certain aspects, gene therapy vectors are provided, for example, gene therapy vectors comprising: (a) a nucleic acid sequence encoding a therapeutic protein, and (b) a nucleic acid sequence encoding a peptide that binds with high affinity to CI-MPR. In some embodiments, the peptide is a variant IGF2(vIGF2) peptide. In some embodiments, the vIGF2 peptide comprises an amino acid sequence at least 90% identical to SEQ ID No. 1 and having at least one substitution at one or more positions selected from the group consisting of: positions 6, 26, 27, 43, 48, 49, 50, 54, 55 and 65 of SEQ ID NO. 1. In some embodiments, the at least one substitution is selected from the group consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R, L55R and K65R of SEQ ID NO. 1. In some embodiments, the vIGF2 peptide comprises at least two substitutions at two or more positions selected from the group consisting of: positions 6, 26, 27, 43, 48, 49, 50, 54 and 55 of SEQ ID NO. 1. In some embodiments, the at least two substitutions are selected from the group consisting ofGroup consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R and L55R of SEQ ID NO. 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at position 1 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-2 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-3 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID NO:1 and substitutions of E6R, Y27L, and K65R. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID NO:1 and substitutions E6R and Y27L. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-5 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-6 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-7 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide has reduced or no affinity for insulin receptor and IGF1R compared to the native IGF2 peptide. In some embodiments, the vIGF2 peptide is capable of promoting uptake of a therapeutic protein into a cell. In some embodiments, the vIGF2 peptide is capable of promoting uptake of a therapeutic protein into lysosomes. In some embodiments, the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder. In some embodiments, the therapeutic protein is a lysosomal enzyme or enzymatically active fragment thereof. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uropathy, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Leehler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippon disease type C Ribopathy, Sanfilippo disease type D, Moquine disease type A, Moquine disease type B, Manoto-Lamy disease, Spirosis, Niemann-pick disease type A, Niemann-pick disease type B, Niemann-pick disease type C1, Niemann-pick disease type C2, Sindler disease type I, and Sindler disease type II. In some embodiments, the lysosomal storage disease is selected from the group consisting of: deficiency of activating factor, GM 2-gangliosidosis; GM 2-gangliosidosis, AB variant; alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe); beta-mannoside storage disease; lysosomal acid lipase deficiency; cystinosis (type of late onset juvenile or juvenile nephropathy; nephropathic nature in infants); christian-dofmann syndrome; neutral lipid storage disease with myopathy; NLSDM; disease of agricultural crops; fabry disease; late onset Fabry disease type II; fabry disease; farber fatty granulomatosis; fucoside storage disorders; galactosialistorage disorder (combined neuraminidase and beta-galactosidase deficiency); gaucher disease; gaucher disease type II; gaucher disease type III; gaucher disease type IIIC; atypical gaucher disease due to sphingolipid activator protein C deficiency; GM 1-gangliosidosis (advanced infantile/juvenile GM 1-gangliosidosis; adult/chronic GM 1-gangliosidosis); globulocyte leukodystrophy, krabbe's disease (late onset infant type; juvenile onset type; adult onset type); atypical krabbe's disease due to sphingolipid activator protein a deficiency; metachromatic leukodystrophy (juvenile; adult); partial sulfaterebroside deficiency; pseudoarylsulfatase a deficiency; metachromatic leukodystrophy due to sphingolipid activator B deficiency; mucopolysaccharide storage disorder: MPS I, heler syndrome; MPS I, heler-schey syndrome; MPS I, schey syndrome; MPS II, hunter syndrome; MPS II, hunter syndrome; sanfilippo syndrome/MPS IIIA type a; sanfilippo syndrome/MPS IIIB type B; sanfilippo syndrome/MPS IIIC type C; sanfilippo syndrome/MPS IIID type D; morquio syndrome, type a/MPS IVA; morquio syndrome, type B/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI marlotte-lamide syndrome; MPS VII sri syndrome; salivary gland disease type I, II mucolipidosis; i-cell disease, Leluo-lolo disease, mucolipidosis Storage disorder II; shampooing malnutrition/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA; type IV mucolipidosis; polythiol esterase deficiency; niemann-pick disease (type B; type C1/chronic neurological form; type C2; type D/Nova scotia); neuronal ceroid lipofuscinosis: CLN6 disease, atypical late infant, late onset variant, early childhood; pasteur/juvenile NCL/CLN3 disease; finnish variant late infant CLN 5; jenski-biziksky disease/late infantile CLN2/TPP1 disease; kuves/adult onset NCL/CLN4 disease (type B); northern epilepsy/variant late infant CLN 8; Santaiwaii-Hardiya/infant CLN1/PPT disease; pompe disease (glycogen storage disease type II); late onset pompe disease; dense bone developmental disorder; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sinderler disease (type III/moderate, variable); qi disease of the Shen; sara disease; infantile asialostorage disease (ISSD); spinal muscular atrophy with progressive myospasm epilepsy (SMAPME); Tay-saxophone/GM 2 gangliosidosis; juvenile onset tay-saxophone disease; late onset tay-saxophone; klissinostian syndrome; lowboy eye-brain-kidney syndrome; type 4J chak-mary-dus disease, CMT 4J; ulis-von syndrome; bilateral temporal occipital multicephalic gyrus (BTOP); x-linked hypercalcemic nephrolithiasis, dent-1; and dengue disease 2, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), Chronic Granulomatous Disease (CGD), and neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is pompe disease. In some embodiments, the genetic disorder is neuronal ceroid lipofuscinosis. In some embodiments, the neuronal ceroid lipofuscinosis is selected from the group consisting of: infant NCL (Santaovri-Hardy's disease), advanced infant NCL (Janski-Bilski disease), Barteng disease, adult NCL (Kofski disease), Finland advanced infant NCL, variant advanced infant NCL, CLN7, CLN8, Turkey advanced infant NCL, NCL 9, and CLN 10. In some embodiments, the therapeutic protein is a soluble lysosomal enzyme. In some embodiments, the therapeutic protein comprises a peptide selected from the group consisting of An enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-2-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., beta 0-L), acetyl-CoA: beta 1-glucosaminyl N-acetyltransferase, glycosaminoglycan beta 3-L-iduronal hydrolase, heparan N-sulfatase, N-acetyl-beta 4-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, alpha-galactosidase-galactosaminyl-N-glucosaminidase, beta-glucosaminidase, glucosylcerase, glucosylceramide-N-sulfatase, glucosylcerase, glucosylcera, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, β 5-N-acetylneuraminidase (sialidase), ganglioside sialidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, battenin, palmitoyl protein thioesterase, and other battten-related proteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein is an alpha-glucosidase or an enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the palmitoyl protein thioesterase is a palmitoyl protein thioesterase 1(PPT1) or 2(PPT 2). In some embodiments, the palmitoyl protein thioesterase is palmitoyl protein thioesterase 1. In some embodiments, the nucleic acid construct further comprises a translation initiation sequence. In some embodiments, the translation initiation sequence comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises the sequence AX 1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of which is any nucleotide. In some embodiments, X1Contains A. In some embodiments, X2Contains G. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence comprises AAGATGA (SEQ ID NO: 29). At one endIn some embodiments, the Kozak sequence comprises a nucleic acid sequence that is at least 85% identical to GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence differs from the sequence of GCAAGATG (SEQ ID NO:44) by one or two nucleotides. In some embodiments, the Kozak sequence comprises GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to CACCATG (SEQ ID NO: 47). In some embodiments, the Kozak sequence differs from the sequence of CACCATG (SEQ ID NO:47) by one or two nucleotides. In some embodiments, the Kozak sequence comprises CACCATG (SEQ ID NO: 47). In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a signal peptide, wherein the signal peptide is capable of increasing secretion of the therapeutic protein compared to the therapeutic protein without the signal peptide. In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a signal peptide, wherein the signal peptide is capable of increasing secretion of the therapeutic protein compared to the therapeutic protein with the native signal peptide. In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding a non-native signal peptide, wherein the non-native signal peptide is capable of increasing secretion of the therapeutic protein compared to a native signal peptide of the therapeutic protein. In some embodiments, the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide. In some embodiments, the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17. In some embodiments, the BiP signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 13-17. In some embodiments, the signal peptide comprises a Gaussia signal peptide. In some embodiments, the Gaussia signal peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32. In some embodiments, the Gaussia signal peptide comprises SEQ ID NO 32. In some embodiments, the vIGF2 nucleic acid sequence is 5' to the nucleic acid sequence encoding the therapeutic protein. In some embodiments, the vIGF2 nucleic acid sequence is 3' to the nucleic acid sequence encoding the therapeutic protein. In some embodiments, the nucleic acid construct further comprises a linker peptide encoding between said vIGF2 nucleotide sequence and said nucleic acid sequence encoding a therapeutic protein And (4) sequencing. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker peptide comprises SEQ ID NO 18-21 or SEQ ID NO 33. In some embodiments, the gene therapy vector is a viral vector. In some embodiments, the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or a herpesvirus vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector comprises an Inverted Terminal Repeat (ITR). In some embodiments, the AAV vector is selected from the group consisting of: AAV1 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector, AAV8 vector, AAV9 vector, AAVrhS vector, AAVrh10 vector, AAVrh33 vector, AAVrh34 vector, AAVrh74 vector, AAV Anc80 vector, aavphp.b vector, AAVhu68 vector and AAV-DJ vector.
A gene therapy vector comprising a nucleic acid construct comprising: (a) a nucleic acid sequence encoding a therapeutic protein, and (b) a nucleic acid sequence encoding a peptide that increases endocytosis of the therapeutic protein. In some embodiments, the peptide that increases endocytosis of the therapeutic protein is a peptide that binds to CI-MPR. In some embodiments, the peptide is a variant IGF2(vIGF2) peptide, HIRMab or TfRMab, or other cell targeting peptide or protein. In some embodiments, the peptide is vIGF 2. In some embodiments, the vIGF2 peptide comprises an amino acid sequence at least 90% identical to SEQ ID No. 1 and having at least one substitution at one or more positions selected from the group consisting of: positions 6, 26, 27, 43, 48, 49, 50, 54, 55 and 65 of SEQ ID NO. 1. In some embodiments, the at least one substitution is selected from the group consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R, L55R and K65R of SEQ ID NO. 1. In some embodiments, the at least one substitution is selected from the group consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R and L55R of SEQ ID NO. 1. In some embodiments, the vI The GF2 peptide comprises at least two substitutions at two or more positions selected from the group consisting of: positions 6, 26, 27, 43, 48, 49, 50, 54 and 55 of SEQ ID NO. 1. In some embodiments, the at least two substitutions are selected from the group consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R and L55R of SEQ ID NO. 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at position 1 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-6 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-2 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-3 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID NO:1 and substitutions of E6R, Y27L, and K65R. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID NO:1 and substitutions E6R and Y27L. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-5 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-6 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-7 of SEQ ID NO: 1. In some embodiments, the vIGF2 peptide has increased specificity for the cation-independent M6P receptor (CI-MPR) compared to the native IGF2 peptide. In some embodiments, the vIGF2 peptide is capable of promoting uptake of a therapeutic protein into lysosomes in cells. In some embodiments, the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder. In some embodiments, the therapeutic protein is a lysosomal enzyme or enzymatically active fragment thereof. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucosaminuria, Barten, cystinosis, Fabry's disease, gaucher type I, gaucher type II, gaucher type III, Pompe disease, Tay-saxophone disease, sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, holler disease, hunter disease, sanfilippo disease type a, sanfilippo disease type B, sanfilippo disease type C, sanfilippo disease type D, morqui disease type a, morqui disease type B, maroto-lamy disease, sri disease, niemann-pick disease type a, niemann-pick disease type B, niemann-pick disease type C1, niemann-pick disease type C2, sindler disease type I, and sindler disease type II. In some embodiments, the lysosomal storage disease is selected from the group consisting of: deficiency of activating factor, GM 2-gangliosidosis; GM 2-gangliosidosis, AB variant; alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe); beta-mannoside storage disease; lysosomal acid lipase deficiency; cystinosis (type of late onset juvenile or juvenile nephropathy; nephropathic nature in infants); christian-dofmann syndrome; neutral lipid storage disease with myopathy; NLSDM; disease of agricultural crops; fabry disease; late onset Fabry disease type II; fabry disease; farber fatty granulomatosis; fucoside storage disorders; galactosialistorage disorder (combined neuraminidase and beta-galactosidase deficiency); gaucher disease; gaucher disease type II; gaucher disease type III; gaucher disease type IIIC; atypical gaucher disease due to sphingolipid activator protein C deficiency; GM 1-gangliosidosis (advanced infantile/juvenile GM 1-gangliosidosis; adult/chronic GM 1-gangliosidosis); globulocyte leukodystrophy, krabbe's disease (late onset infant type; juvenile onset type; adult onset type); atypical krabbe's disease due to sphingolipid activator protein a deficiency; metachromatic leukodystrophy (juvenile; adult); partial sulfaterebroside deficiency; pseudoarylsulfatase a deficiency; metachromatic leukodystrophy due to sphingolipid activator B deficiency; mucopolysaccharide storage disorder: MPS I, heler syndrome; MPS I, heler-schey syndrome; MPS I, schey syndrome; MPS II, hunter syndrome; MPS II, hunter syndrome; sanfilippo syndrome/MPS IIIA type a; sanfilippo syndrome/MPS IIIB type B; sanfilippo syndrome/MPS IIIC type C; stiff wave syndrome D MPS IIID; morquio syndrome, type a/MPS IVA; morquio syndrome, type B/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI marlotte-lamide syndrome; MPS VII sri syndrome; salivary gland disease type I, II mucolipidosis; i-cell disease, Leluo disease, mucolipidosis II; shampooing malnutrition/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA; type IV mucolipidosis; polythiol esterase deficiency; niemann-pick disease (type B; type C1/chronic neurological form; type C2; type D/Nova scotia); neuronal ceroid lipofuscinosis: CLN6 disease, atypical late infant, late onset variant, early childhood; pasteur/juvenile NCL/CLN3 disease; finnish variant late infant CLN 5; jenski-biziksky disease/late infantile CLN2/TPP1 disease; kuves/adult onset NCL/CLN4 disease (type B); northern epilepsy/variant late infant CLN 8; Santaiwaii-Hardiya/infant CLN1/PPT disease; pompe disease (glycogen storage disease type II); late onset pompe disease; dense bone developmental disorder; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sinderler disease (type III/moderate, variable); qi disease of the Shen; sara disease; infantile asialostorage disease (ISSD); spinal muscular atrophy with progressive myospasm epilepsy (SMAPME); Tay-saxophone/GM 2 gangliosidosis; juvenile onset tay-saxophone disease; late onset tay-saxophone; klissinostian syndrome; lowboy eye-brain-kidney syndrome; type 4J chak-mary-dus disease, CMT 4J; ulis-von syndrome; bilateral temporal occipital multicephalic gyrus (BTOP); x-linked hypercalcemic nephrolithiasis, dent-1; and dengue disease 2, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), Chronic Granulomatous Disease (CGD), and neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is pompe disease. In some embodiments, the genetic disorder is neuronal ceroid lipofuscinosis. In some embodiments, the neuronal ceroid lipofuscinosis is selected from the group consisting of: infantile NCL (Santaooli-Hardy disease), advanced NCL (Janski-Bierwolski disease), Barteng disease, adult NCL (Kofski disease), Finnish late infant NCL, variant late infant NCL, CLN7, CLN8, Turkey late infant NCL, NCL 9 and CLN 10. In some embodiments, the therapeutic protein is a soluble lysosomal enzyme or enzymatically active fragment thereof. In some embodiments, the therapeutic protein comprises an enzyme selected from the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-2-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., beta 0-L), acetyl-CoA: beta 1-glucosaminyl N-acetyltransferase, glycosaminoglycan beta 3-L-iduronal hydrolase, heparan N-sulfatase, N-acetyl-beta 4-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, alpha-galactosidase-galactosaminyl-N-glucosaminidase, beta-glucosaminidase, glucosylcerase, glucosylceramide-N-sulfatase, glucosylcerase, glucosylcera, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, β 5-N-acetylneuraminidase (sialidase), ganglioside sialidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, battenin, palmitoyl protein thioesterase, and other battten-related proteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein is an alpha-glucosidase, or an enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the palmitoyl protein thioesterase is a palmitoyl protein thioesterase 1(PPT1) or 2(PPT 2). In some embodiments, the palmitoyl protein thioesterase is palmitoyl protein thioesterase 1. In some embodiments, the nucleic acid construct further comprises a translation initiation sequence. In some embodiments, the translation initiation sequence comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises the sequence AX 1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of which is any nucleotide. In some casesIn the embodiment, X1Contains A. In some embodiments, X2Contains G. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence comprises AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence differs from the sequence of GCAAGATG (SEQ ID NO:44) by one or two nucleotides. In some embodiments, the Kozak sequence comprises GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to CACCATG (SEQ ID NO: 47). In some embodiments, the Kozak sequence differs from the sequence of CACCATG (SEQ ID NO:47) by one or two nucleotides. In some embodiments, the Kozak sequence comprises CACCATG (SEQ ID NO: 47). In some embodiments, the nucleic acid construct further comprises a signal nucleic acid sequence encoding a signal peptide, wherein the signal peptide is capable of increasing secretion of the therapeutic protein compared to the therapeutic protein without the signal peptide. In some embodiments, the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide. In some embodiments, the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17. In some embodiments, the BiP signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 13-17. In some embodiments, the signal peptide comprises a Gaussia signal peptide. In some embodiments, the Gaussia signal peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32. In some embodiments, the Gaussia signal peptide comprises SEQ ID NO 32. In some embodiments, the vIGF2 nucleic acid sequence is 5' to the nucleic acid sequence encoding the therapeutic protein. In some embodiments, the vIGF2 nucleic acid sequence is 3' to the nucleic acid sequence encoding the therapeutic protein. In some embodiments, the nucleic acid construct further comprises a linker sequence encoding a linker peptide between said vIGF2 nucleotide sequence and said nucleic acid sequence encoding a therapeutic protein. In some embodiments, the linker is composed of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids Amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker peptide comprises SEQ ID NO 18-21 or SEQ ID NO 33. In some embodiments, the gene therapy vector is a viral vector. In some embodiments, the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or a herpesvirus vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector comprises an Inverted Terminal Repeat (ITR). In some embodiments, the AAV vector is selected from the group consisting of: AAV1 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector, AAV8 vector, AAV9 vector, AAVrhS vector, AAVrh10 vector, AAVrh33 vector, AAVrh34 vector, AAVrh74 vector, AAV Anc80 vector, aavphp.b vector, AAVhu68 vector and AAV-DJ vector.
In a further aspect, there is provided a pharmaceutical composition comprising (i) a therapeutically effective amount of any one of the gene therapy vectors herein, and (ii) a pharmaceutically acceptable carrier or excipient. In some embodiments, the carrier or excipient comprises a non-ionic, low-permeability compound, buffer, polymer, salt, or combination thereof.
In other aspects, methods are provided for treating a genetic disorder, the methods comprising administering to a subject in need thereof any one of the gene therapy vectors provided herein or any one of the pharmaceutical compositions provided herein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine diabetes, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, MuldoHoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hewler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Moquini disease type A, Moquine disease type B, Maruto-Lami disease, Sprius disease, Niemann-pick disease type A, Niemann-pick disease type B, Niemann-pick disease type C1, Niemann-pick disease type C2, Ocimir disease type I and Ocimier disease type II. In some embodiments, the lysosomal storage disease is selected from the group consisting of: deficiency of activating factor, GM 2-gangliosidosis; GM 2-gangliosidosis, AB variant; alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe); beta-mannoside storage disease; lysosomal acid lipase deficiency; cystinosis (type of late onset juvenile or juvenile nephropathy; nephropathic nature in infants); christian-dofmann syndrome; neutral lipid storage disease with myopathy; NLSDM; disease of agricultural crops; fabry disease; late onset Fabry disease type II; fabry disease; farber fatty granulomatosis; fucoside storage disorders; galactosialistorage disorder (combined neuraminidase and beta-galactosidase deficiency); gaucher disease; gaucher disease type II; gaucher disease type III; gaucher disease type IIIC; atypical gaucher disease due to sphingolipid activator protein C deficiency; GM 1-gangliosidosis (advanced infantile/juvenile GM 1-gangliosidosis; adult/chronic GM 1-gangliosidosis); globulocyte leukodystrophy, krabbe's disease (late onset infant type; juvenile onset type; adult onset type); atypical krabbe's disease due to sphingolipid activator protein a deficiency; metachromatic leukodystrophy (juvenile; adult); partial sulfaterebroside deficiency; pseudoarylsulfatase a deficiency; metachromatic leukodystrophy due to sphingolipid activator B deficiency; mucopolysaccharide storage disorder: MPS I, heler syndrome; MPS I, heler-schey syndrome; MPS I, schey syndrome; MPS II, hunter syndrome; MPS II, hunter syndrome; sanfilippo syndrome/MPS IIIA type a; sanfilippo syndrome/MPS IIIB type B; sanfilippo syndrome/MPS IIIC type C; sanfilippo syndrome/MPS IIID type D; morquio syndrome, type a/MPS IVA; morquio syndrome, type B/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI marlotte-lamide syndrome; MPS VII sri syndrome; salivary gland disease type I, II mucolipidosis; i-cell disease, Leluo disease, mucolipidosis II; shampooing malnutrition/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA; type IV mucolipidosis; polythiol esterase deficiency; niemann-pick disease (type B; type C1/chronic neurological form; type C2; type D/Nova scotia); neuronal ceroid lipofuscinosis: CLN6 disease, atypical late infant, late onset variant, early childhood; pasteur/juvenile NCL/CLN3 disease; finnish variant late infant CLN 5; jenski-biziksky disease/late infantile CLN2/TPP1 disease; kuves/adult onset NCL/CLN4 disease (type B); northern epilepsy/variant late infant CLN 8; Santaiwaii-Hardiya/infant CLN1/PPT disease; pompe disease (glycogen storage disease type II); late onset pompe disease; dense bone developmental disorder; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sinderler disease (type III/moderate, variable); qi disease of the Shen; sara disease; infantile asialostorage disease (ISSD); spinal muscular atrophy with progressive myospasm epilepsy (SMAPME); Tay-saxophone/GM 2 gangliosidosis; juvenile onset tay-saxophone disease; late onset tay-saxophone; klissinostian syndrome; lowboy eye-brain-kidney syndrome; type 4J chak-mary-dus disease, CMT 4J; ulis-von syndrome; bilateral temporal occipital multicephalic gyrus (BTOP); x-linked hypercalcemic nephrolithiasis, dent-1; and dengue disease 2, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), Chronic Granulomatous Disease (CGD), CDKL5 deficiency, and neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is pompe disease. In some embodiments, the genetic disorder is neuronal ceroid lipofuscinosis. In some embodiments, the neuronal ceroid lipofuscinosis is selected from the group consisting of: infant NCL (Santaovri-Hardy's disease), advanced infant NCL (Janski-Bilski disease), Barteng disease, adult NCL (Kofski disease), Finland advanced infant NCL, variant advanced infant NCL, CLN7, CLN8, Turkey advanced infant NCL, NCL 9, and CLN 10. In some embodiments, administration is intrathecal, intraocular, intravitreal, transretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intracerebroventricular, intraparenchymal, intracameral, subcutaneous, or a combination thereof. In some embodiments, administration is performed intrathecally. In some embodiments, administration is intraocular, intravitreal, or transretinal.
In a further aspect, there is provided a pharmaceutical composition comprising any one of the gene therapy vectors herein and a pharmaceutically acceptable carrier or excipient for use in the treatment of a genetic disorder. In other aspects, pharmaceutical compositions are provided comprising any one of the gene therapy vectors herein and a pharmaceutically acceptable carrier or excipient for use in the preparation of a medicament for the treatment of a genetic disorder. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine diabetes, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, MuldoHoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hewler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Moquine disease type A, Moquine disease type B, Maruto-Lami disease, Sprius disease, Niemann-pick disease type A, Niemann-pick disease type B, Niemann-pick disease type C1, Niemann-pick disease type C2, Sinidel disease type I and Sindler disease type II. In some embodiments, the lysosomal storage disease is selected from the group consisting of: deficiency of activating factor, GM 2-gangliosidosis; GM 2-gangliosidosis, AB variant; alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe); beta-mannoside storage disease; lysosomal acid lipase deficiency; cystinosis (type of late onset juvenile or juvenile nephropathy; nephropathic nature in infants); christian-dofmann syndrome; neutral lipid storage disease with myopathy; NLSDM; disease of agricultural crops; fabry disease; late onset Fabry disease type II; fabry disease; farber fatty granulomatosis; fucoside storage disorders; galactosialistorage disorder (combined neuraminidase and beta-galactosidase deficiency); gaucher disease; gaucher disease type II; gaucher disease type III; gaucher disease type IIIC; atypical gaucher disease due to sphingolipid activator protein C deficiency; GM 1-gangliosidosis (advanced infantile/juvenile GM 1-gangliosidosis; adult/chronic GM 1-gangliosidosis); globulocyte leukodystrophy, krabbe's disease (late onset infant type; juvenile onset type; adult onset type); atypical krabbe's disease due to sphingolipid activator protein a deficiency; metachromatic leukodystrophy (juvenile; adult); partial sulfaterebroside deficiency; pseudoarylsulfatase a deficiency; metachromatic leukodystrophy due to sphingolipid activator B deficiency; mucopolysaccharide storage disorder: MPS I, heler syndrome; MPS I, heler-schey syndrome; MPS I, schey syndrome; MPS II, hunter syndrome; MPS II, hunter syndrome; sanfilippo syndrome/MPS IIIA type a; sanfilippo syndrome/MPS IIIB type B; sanfilippo syndrome/MPS IIIC type C; sanfilippo syndrome/MPS IIID type D; morquio syndrome, type a/MPS IVA; morquio syndrome, type B/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI marlotte-lamide syndrome; MPS VII sri syndrome; salivary gland disease type I, II mucolipidosis; i-cell disease, Leluo disease, mucolipidosis II; shampooing malnutrition/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA; type IV mucolipidosis; polythiol esterase deficiency; niemann-pick disease (type B; type C1/chronic neurological form; type C2; type D/Nova scotia); neuronal ceroid lipofuscinosis: CLN6 disease, atypical late infant, late onset variant, early childhood; pasteur/juvenile NCL/CLN3 disease; finnish variant late infant CLN 5; jenski-biziksky disease/late infantile CLN2/TPP1 disease; kuves/adult onset NCL/CLN4 disease (type B); northern epilepsy/variant late infant CLN 8; Santaiwaii-Hardiya/infant CLN1/PPT disease; pompe disease (glycogen storage disease type II); late onset pompe disease; dense bone developmental disorder; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sinderler disease (type III/moderate, variable); qi disease of the Shen; sara disease; infantile asialostorage disease (ISSD); spinal muscular atrophy with progressive myospasm epilepsy (SMAPME); Tay-saxophone/GM 2 gangliosidosis; juvenile onset tay-saxophone disease; late onset tay-saxophone; klissinostian syndrome; lowboy eye-brain-kidney syndrome; type 4J chak-mary-dus disease, CMT 4J; ulis-von syndrome; bilateral temporal occipital multicephalic gyrus (BTOP); x-linked hypercalcemic nephrolithiasis, dent-1; and dengue disease 2, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), Chronic Granulomatous Disease (CGD), CDKL5 deficiency, and neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is pompe disease. In some embodiments, the genetic disorder is neuronal ceroid lipofuscinosis. In some embodiments, the neuronal ceroid lipofuscinosis is selected from the group consisting of: infant NCL (Santaovri-Hardy's disease), advanced infant NCL (Janski-Bilski disease), Barteng disease, adult NCL (Kofski disease), Finland advanced infant NCL, variant advanced infant NCL, CLN7, CLN8, Turkey advanced infant NCL, NCL 9, and CLN 10. In some embodiments, the composition is formulated for intrathecal, intraocular, intravitreal, transretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, ocular, or subcutaneous administration. In some embodiments, the composition is formulated for intrathecal administration. In some embodiments, the composition is formulated for intrathecal administration for the treatment of a neurodegenerative disorder. In some embodiments, the composition is formulated for ocular, intravitreal, or transretinal administration.
Provided herein are gene therapy vectors comprising a nucleic acid construct encoding a polypeptide comprising: (a) a therapeutic protein; (b) a peptide that binds with high affinity to cation-independent mannose 6-phosphate (M6P) receptor (CI-MPR); and (c) a linker between the therapeutic protein and the CI-MPR binding peptide. In some embodiments, the peptide is a variant IGF2(vIGF2) peptide. In some embodiments, the vIGF2 peptide comprises an amino acid sequence at least 90% identical to SEQ ID No. 1 and having at least one substitution at one or more positions selected from the group consisting of: positions 6, 26, 27, 43, 48, 49, 50, 54, 55 and 65 of SEQ ID NO. 1. In some embodiments, the at least one substitution is selected from the group consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R, L55R and K65R of SEQ ID NO. 1. In some embodiments, the vIGF2 peptide comprises at least two substitutions at two or more positions selected from the group consisting of: 1, positions 6, 26, 27, 43, 48, 49, 50, 54, 55, 65. In some embodiments, the at least two substitutions are selected from the group consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R, L55R, K65R of SEQ ID NO. 1. In some embodiments, the vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID NO: 1. In some embodiments, wherein the vIGF2 peptide has reduced affinity for insulin receptor and IGF1R compared to the native IGF2 peptide. In some embodiments, the vIGF2 peptide is capable of promoting uptake of the therapeutic protein into a cell. In some embodiments, the vIGF2 peptide is capable of promoting uptake of the therapeutic protein into lysosomes. In some embodiments, the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hewlett-packard disease, Hunter disease, sanfilippo disease type A, sanfilippo disease type B, sanfilippo disease type C, sanfilippo disease type D, moquinase type A, moquinase type B, maratoto-lamic disease, sjgren disease, niemann-pick disease type A, niemann-pick disease type B, niemann-pick disease type C1, niemann-pick disease type C2, sinderler disease type I, sinderler disease type II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), Chronic Granulomatosis (CGD), and neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is pompe disease. In some embodiments, the genetic disorder is CLN1 disease. In some embodiments, the therapeutic protein comprises a soluble lysosomal enzyme or enzymatically active fragment thereof. In some embodiments, the therapeutic protein comprises a lysosomal enzyme or enzymatically active fragment thereof, wherein the lysosomal enzyme is selected from the group consisting of: alpha-galactosidase A, beta-glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan alpha-L-iduronate hydrolase, iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, palmitoyl protein thioesterase, cyclin-dependent kinase-like 5, and alpha-glucosidase. In some embodiments, the therapeutic protein is an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is a palmitoyl protein thioesterase or an enzymatically active fragment thereof. In some embodiments, the therapeutic protein is palmitoyl protein thioesterase-1 or an enzymatically active fragment thereof. In some embodiments, the nucleic acid construct further comprises a translation initiation sequence. In some embodiments, the translation initiation sequence comprises a Kozak sequence. In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a signal peptide, wherein the signal peptide is capable of increasing secretion of a therapeutic protein compared to the therapeutic protein without the signal peptide. In some embodiments, the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide. In some embodiments, the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17. In some embodiments, the vIGF2 peptide comprises the sequence of SEQ ID No. 31. In some embodiments, the construct comprises SEQ ID NO 36. In some embodiments, the polypeptide comprises SEQ ID NO 23. In some embodiments, the construct comprises SEQ ID NO 38. In some embodiments, vIGF2 is at the N-terminus of the polypeptide. In some embodiments, vIGF2 is C-terminal to the polypeptide. In some embodiments, the linker peptide comprises SEQ ID NO 18-21 or SEQ ID NO 33. In some embodiments, the gene therapy vector is a viral vector selected from the group consisting of: adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, poxvirus vectors, vaccinia virus vectors, adenoviral vectors, and herpesvirus vectors.
In certain aspects, fusion proteins, e.g., fusion proteins comprising a variant IGF2 peptide and a therapeutic protein, are provided. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37, also referred to herein as "2 GS") or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein is encoded by a nucleic acid comprising a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the fusion protein is encoded by a nucleic acid comprising a Kozak sequence.
In a further aspect, a fusion protein is provided comprising a signal peptide and a therapeutic protein, wherein the signal peptide is post-translationally removed upon secretion from a cell. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the fusion protein is encoded by a nucleic acid comprising a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the fusion protein is encoded by a nucleic acid comprising a Kozak sequence.
In other aspects, a nucleic acid sequence encoding a fusion protein comprising a therapeutic protein is provided, wherein the fusion protein is encoded by a nucleic acid comprising a cricket palsy virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the fusion protein is encoded by a nucleic acid comprising a Kozak sequence.
In a further aspect, a fusion protein comprising a therapeutic protein is provided, wherein the fusion protein is encoded by a nucleic acid comprising a Kozak sequence. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the fusion protein is encoded by a nucleic acid comprising a cricket paralysis virus internal ribosome entry sequence (CrPV IRES).
In a further aspect, there is provided a nucleic acid encoding a fusion protein, for example a nucleic acid encoding a fusion protein comprising a variant IGF2 peptide and a therapeutic protein. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus.
In other aspects, nucleic acids encoding fusion proteins comprising a signal peptide and a therapeutic protein are provided. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus.
In a further aspect, there is provided a nucleic acid encoding a fusion protein comprising a therapeutic protein, wherein the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus.
In other aspects, there is provided a nucleic acid encoding a fusion protein comprising a therapeutic protein, wherein the nucleic acid further comprises a Kozak sequence. In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminide N-acetyltransferase, glycosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminide (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetyl-alpha-D-glucosaminide (NAGLU), N-acetyl-glucosaminyl-2-sulfatase, alpha-L-glucosaminyl-6-sulfatase, N-acetyl-sulfatase, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminide (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus.
In a further aspect, there is provided a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a variant IGF2 peptide and a therapeutic protein; and (b) a buffer or excipient suitable for gene therapy. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein.
In a further aspect, there is provided a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a signal peptide and a therapeutic protein; and (b) a buffer or excipient suitable for gene therapy. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein.
In other aspects, compositions are provided, comprising: (a) a nucleic acid encoding a fusion protein comprising a therapeutic protein; and (b) a buffer or excipient suitable for gene therapy, wherein the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein.
In other aspects, compositions are provided, comprising: (a) a nucleic acid encoding a fusion protein comprising a therapeutic protein; and (b) a buffer or excipient suitable for gene therapy, wherein the nucleic acid further comprises a Kozak sequence. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein.
In a further aspect, there is provided a method of treating a genetic disorder in an individual, the method comprising administering a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a variant IGF2 peptide and a therapeutic protein; and (b) a buffer or excipient suitable for gene therapy. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease A, san Francisella disease B, san Francisella disease C, san Francisella disease D, morqui disease A, morqui disease B, Maruo-lamide disease, Spirosis, Niemann-pick disease A, Niemann-pick disease B, Niemann-pick disease C1, Niemann-pick disease C2, Sindler disease I, Sindler disease II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), and Chronic Granulomatosis (CGD). In some embodiments, cells from an individual are treated ex vivo, and the cells are administered to the individual after ex vivo treatment.
In a further aspect, there is provided a method of treating a genetic disorder in an individual, the method comprising administering a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a signal peptide and a therapeutic protein; and (b) a buffer or excipient suitable for gene therapy. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease A, san Francisella disease B, san Francisella disease C, san Francisella disease D, morqui disease A, morqui disease B, Maruo-lamide disease, Spirosis, Niemann-pick disease A, Niemann-pick disease B, Niemann-pick disease C1, Niemann-pick disease C2, Sindler disease I, Sindler disease II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), and Chronic Granulomatosis (CGD). In some embodiments, cells from an individual are treated ex vivo, and the cells are administered to the individual after ex vivo treatment.
In other aspects, there is provided a method of treating a genetic disorder in an individual, the method comprising administering a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a therapeutic protein and a targeting peptide; and (b) a buffer or excipient suitable for gene therapy, wherein the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease A, san Francisella disease B, san Francisella disease C, san Francisella disease D, morqui disease A, morqui disease B, Maruo-lamide disease, Spirosis, Niemann-pick disease A, Niemann-pick disease B, Niemann-pick disease C1, Niemann-pick disease C2, Sindler disease I, Sindler disease II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), and Chronic Granulomatosis (CGD). In some embodiments, cells from an individual are treated ex vivo, and the cells are administered to the individual after ex vivo treatment.
In a further aspect, there is provided a method of treating a genetic disorder in an individual, the method comprising administering a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a therapeutic protein; and (b) a buffer or excipient suitable for gene therapy, wherein the nucleic acid further comprises a Kozak sequence. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease A, san Francisella disease B, san Francisella disease C, san Francisella disease D, morqui disease A, morqui disease B, Maruo-lamide disease, Spirosis, Niemann-pick disease A, Niemann-pick disease B, Niemann-pick disease C1, Niemann-pick disease C2, Sindler disease I, Sindler disease II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), and Chronic Granulomatosis (CGD). In some embodiments, cells from an individual are treated ex vivo, and the cells are administered to the individual after ex vivo treatment.
In other aspects, methods of treating a genetic disorder in an individual are provided, the methods comprising administering a cell comprising a nucleic acid encoding a fusion protein comprising a variant IGF2 peptide and a therapeutic protein. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease A, san Francisella disease B, san Francisella disease C, san Francisella disease D, morqui disease A, morqui disease B, Maruo-lamide disease, Spirosis, Niemann-pick disease A, Niemann-pick disease B, Niemann-pick disease C1, Niemann-pick disease C2, Sindler disease I, Sindler disease II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), and Chronic Granulomatosis (CGD). In some embodiments, the cell is derived from an individual.
In a further aspect, there is provided a method of treating a genetic disorder in an individual, the method comprising administering a cell comprising a nucleic acid encoding a fusion protein comprising a signal peptide and a therapeutic protein. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease A, san Francisella disease B, san Francisella disease C, san Francisella disease D, morqui disease A, morqui disease B, Maruo-lamide disease, Spirosis, Niemann-pick disease A, Niemann-pick disease B, Niemann-pick disease C1, Niemann-pick disease C2, Sindler disease I, Sindler disease II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), and Chronic Granulomatosis (CGD). In some embodiments, the cell is derived from an individual.
In a further aspect, there is provided a method of treating a genetic disorder in an individual, the method comprising administering a cell comprising a nucleic acid encoding a fusion protein comprising a therapeutic protein, wherein the nucleic acid further comprises a cricket palsy virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient suitable for gene therapy comprises a liposome, a nanoparticle, or a cell penetrating peptide. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease A, san Francisella disease B, san Francisella disease C, san Francisella disease D, morqui disease A, morqui disease B, Maruo-lamide disease, Spirosis, Niemann-pick disease A, Niemann-pick disease B, Niemann-pick disease C1, Niemann-pick disease C2, Sindler disease I, Sindler disease II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), and Chronic Granulomatosis (CGD). In some embodiments, the cell is derived from an individual.
In other aspects, methods of treating a genetic disorder in an individual are provided, the methods comprising administering a cell comprising a nucleic acid encoding a fusion protein comprising a therapeutic protein, wherein the nucleic acid further comprises a Kozak sequence. In some embodiments, the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES). In some embodiments, the fusion protein further comprises a linker. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 7, 8, 9, 10, 11, 12, or 13 amino acids. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGG (SEQ ID NO:18), GGGGS (SEQ ID NO:19), GGGSGGGGS (SEQ ID NO:20), GGGGSGGGS (SEQ ID NO:21), GGGGSGGGGS (SEQ ID NO:37), or GGSGSGSTS (SEQ ID NO: 33). In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises a Binding Immunoglobulin Protein (BiP) signal peptide. In some embodiments, the fusion protein further comprises a variant IGF2 peptide. In some embodiments, the therapeutic protein comprises at least one enzyme of the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof. In some embodiments, the therapeutic protein comprises an alpha-glucosidase or enzymatically active fragment thereof. In some embodiments, the therapeutic protein is N-acetyl- α -D-glucosaminidase (NAGLU). In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is contained within a viral vector. In some embodiments, the viral vector comprises a retrovirus, adenovirus, adeno-associated virus, lentivirus, or herpes virus. In some embodiments, the buffer or excipient is suitable for gene therapy. In some embodiments, the buffer or excipient suitable for gene therapy comprises a viral coat protein. In some embodiments, the viral coat protein is selected from the group consisting of: vesicular stomatitis virus coat protein, adenovirus coat protein, adeno-associated virus coat protein, murine leukemia virus coat protein, HIV coat protein, and influenza virus coat protein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease A, san Francisella disease B, san Francisella disease C, san Francisella disease D, morqui disease A, morqui disease B, Maruo-lamide disease, Spirosis, Niemann-pick disease A, Niemann-pick disease B, Niemann-pick disease C1, Niemann-pick disease C2, Sindler disease I, Sindler disease II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), and Chronic Granulomatosis (CGD). In some embodiments, the cell is derived from an individual.
Also provided herein is a fusion protein comprising a native signal peptide, an ER protein cleavage domain, a variant IGF2 peptide, and an alpha-glucosidase lacking its native signal peptide, wherein the fusion protein is encoded by a nucleic acid comprising a Kozak sequence.
Further provided herein is a fusion protein comprising a Binding Immunoglobulin Protein (BiP) signal peptide, a variant IGF2 peptide, and an alpha-glucosidase, wherein the fusion protein is encoded by a nucleic acid comprising a Kozak sequence.
Further provided herein is a fusion protein comprising an immunoglobulin protein (BiP) binding signal peptide, a variant IGF2 peptide, and an alpha-glucosidase lacking its native signal peptide, wherein the fusion protein is encoded by a nucleic acid comprising a cricket paralysis virus internal ribosome entry sequence (CrPV IRES).
Further provided herein is a nucleic acid encoding a fusion protein comprising a native signal peptide, an ER protein cleavage domain, a variant IGF2 peptide, and an alpha-glucosidase lacking its native signal peptide.
Further provided herein is a nucleic acid encoding a fusion protein comprising an immunoglobulin protein (BiP) binding signal peptide, a variant IGF2 peptide, and an alpha-glucosidase lacking its native signal peptide, wherein the nucleic acid further comprises a Kozak sequence.
Further provided herein is a nucleic acid encoding a fusion protein comprising a variant IGF2 peptide and an alpha-glucosidase, wherein the nucleic acid further comprises a cricket paralysis virus internal ribosome entry sequence (CrPV IRES).
Further provided herein is a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a native signal peptide, an ER protein cleavage domain, a variant IGF2 peptide, and an alpha-glucosidase lacking its native signal peptide; and (b) a buffer or excipient suitable for gene therapy.
Further provided herein is a composition comprising: (a) a nucleic acid encoding a fusion protein comprising an immunoglobulin protein (BiP) -binding signal peptide, a variant IGF2 peptide, and an alpha-glucosidase lacking its native signal peptide, wherein the nucleic acid further comprises a Kozak sequence; and (b) a buffer or excipient suitable for gene therapy.
Further provided herein is a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a variant IGF2 peptide and an alpha-glucosidase, wherein the nucleic acid further comprises a cricket palsy virus internal ribosome entry sequence (CrPV IRES); and (b) a buffer or excipient suitable for gene therapy.
Further provided herein is a method of treating pompe disease in an individual, the method comprising administering a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a native signal peptide, an ER protein cleavage domain, a variant IGF2 peptide, and an alpha-glucosidase lacking its native signal peptide; and (b) a buffer or excipient suitable for gene therapy.
Further provided herein is a method of treating pompe disease in an individual, the method comprising administering a composition comprising: (a) a nucleic acid encoding a fusion protein comprising an immunoglobulin protein (BiP) -binding signal peptide, a variant IGF2 peptide, and an alpha-glucosidase lacking its native signal peptide, wherein the nucleic acid further comprises a Kozak sequence; and (b) a buffer or excipient suitable for gene therapy.
Further provided herein is a method of treating pompe disease in an individual, the method comprising administering a composition comprising: (a) a nucleic acid encoding a fusion protein comprising a variant IGF2 peptide and an alpha-glucosidase, wherein the nucleic acid further comprises a cricket palsy virus internal ribosome entry sequence (CrPV IRES); and (b) a buffer or excipient suitable for gene therapy.
Further provided herein is a method of treating pompe disease in an individual comprising administering a cell comprising a nucleic acid encoding a fusion protein comprising a native signal peptide, an ER protein cleavage domain, a variant IGF2 peptide, and an alpha-glucosidase lacking its native signal peptide.
Further provided herein is a method of treating pompe disease in an individual, the method comprising administering a cell comprising a nucleic acid encoding a fusion protein comprising a Binding Immunoglobulin Protein (BiP) signal peptide, a variant IGF2 peptide, and an alpha-glucosidase or enzymatically active fragment thereof, wherein the nucleic acid further comprises a Kozak sequence.
Further provided herein is a method of treating pompe disease in an individual, the method comprising administering a cell comprising a nucleic acid encoding a fusion protein comprising a variant IGF2 peptide and an alpha-glucosidase, wherein the nucleic acid further comprises a cricket palsy virus internal ribosome entry sequence (CrPV IRES).
Is incorporated by reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Drawings
The patent application document contains at least one drawing executed in color. Copies of this patent application with color drawing(s) will be provided by the office upon request and payment of the necessary fee. An understanding of the features and advantages of the present disclosure will be obtained with reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
figure 1 shows GAA activity of the arabinosidase-alpha rhGAA with and without M6P. FIG. 1 shows the scale of a commercial ERT that can incorporate CI-MPR. The first peak is rhGAA that lacks any M6P-containing glycans and therefore cannot be taken up and delivered to lysosomes. The second peak is a fraction containing at least one phosphorylated glycan and having the potential to be taken up by cells and delivered to lysosomes for glycogenolysis.
Figure 2 shows the structure of CI-MPR comprising different binding domains of IGF2 and mono-and di-phosphorylated oligosaccharides.
Figure 3 shows the sequence and structure of mature human IGF2 peptide. Site-specific amino acid substitutions are proposed to affect the binding of other receptors.
Figure 4 shows the binding of wild-type IGF2(wtIGF2) peptide to CI-MPR as measured by surface plasmon resonance.
Figure 5 shows the binding of variant IGF2(vIGF2) peptide bound to CI-MPR as measured by surface plasmon resonance.
Figure 6 shows the addition of vIGF2 to glucosidase a to increase the benefit of binding to IGF 2/CI-MPR.
Figure 7 shows the benefit of adding vIGF2 to recombinant human N-acetyl- α -D-glucamine enzyme (rhNAGLU) to increase binding to IGF 2/CI-MPR.
Figure 8 shows the binding of wild-type human IGF2 to the insulin receptor.
Fig. 9 shows no detectable binding of vIGF2 to the insulin receptor.
Figure 10 shows the binding of wild-type IGF2 to insulin-like growth factor 1 receptor.
Figure 11 shows that vIGF2 peptide has reduced binding to insulin-like growth factor 1 receptor compared to wild-type IGF 2.
FIG. 12 shows two examples of gene therapy expression cassettes encoding native hGAA and engineered hGAA. Native hGAA is poorly phosphorylated, resulting in poor CIMPR binding and cellular uptake. The engineered hGAA has the addition of an element for improved cimr binding (vIGF2), a 2GS linker that reduces the steric hindrance of vIGF2-GAA protein to cimr, and a BiP signal peptide for improved secretion.
Figure 13 shows western blots of PPT1 from cells expressing recombinant human PPT1(PPT1-1), recombinant human PPT1 with a vIGF2 targeting domain (PPT1-2), and recombinant human PPT1 with a vIGF2 targeting domain and a BiP signal sequence (PPT 1-29).
Figure 14 shows binding of PPT1 construct to CI-MPR.
FIG. 15 shows GAA activity in conditioned media of CHO cells expressing engineered or native hGAA.
Figure 16 shows the study design of a 4-week gene therapy mouse study in GAA knockout mice.
Figure 17 shows GAA plasma activity in untreated wild type ("normal") mice or GAA knockout mice treated with gene therapy vectors or vehicles as indicated.
Figure 18 shows GAA levels measured in untreated wild type ("normal") mice or GAA knockout mice treated with gene therapy vectors or vehicles as indicated.
Figure 19 shows cell surface receptor binding of rhGAA from plasma samples obtained from treated mice as indicated.
Figure 20 shows GAA activity and tetrasaccharide histopathology scores of the tibialis anterior of untreated wild type ("normal") mice or GAA knockout mice treated with gene therapy vectors or vehicles as indicated.
Figure 21 shows glycogen PAS from the tibialis anterior as indicated from untreated wild type mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 22 shows hGAA immunohistochemistry as indicated for tibialis anterior from untreated wild type mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 23 shows histopathological scores of brain GAA activity, brain glycogen and spinal cord glycogen as indicated from brain and spinal cord of untreated wild type ("normal") mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 24 shows glycogen PAS from brain of untreated wild type mice or GAA knockout mice treated with gene therapy vectors or vehicles as indicated.
Figure 25 shows hGAA immunohistochemistry for brainstem and choroid plexus as indicated from untreated wild type ("normal") mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 26 shows glycogen PAS from spinal cord of untreated wild type mice or GAA knockout mice treated with gene therapy vectors or vehicles as indicated.
Figure 27 shows hGAA immunohistochemistry as indicated from spinal cord of untreated wild type mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 28 shows quadriceps GAA activity and glycogen histopathology scores as indicated from untreated wild type ("normal") mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 29 shows glycogen luxol/PAS as indicated from quadriceps of untreated wild type mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 30 shows hGAA immunohistochemistry for quadriceps as indicated from untreated wild type mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 31 shows triceps GAA activity and histopathological scores as indicated from untreated wild type ("normal") mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 32 shows glycogen luxol/PAS as indicated from triceps of untreated wild type mice or GAA knockout mice treated with gene therapy vectors or vehicles.
Figure 33 shows hGAA immunohistochemistry for triceps from untreated wild type mice or GAA knockout mice treated with gene therapy vectors or vehicles as indicated.
Detailed Description
Gene therapy of single gene genetic disorders provides a potential single treatment for diseases and disorders, some of which have destructive symptoms that may occur early in life and sometimes lead to lifelong disability. Neurogenetic disorders, such as lysosomal storage diseases, are typically treated with enzyme replacement therapy, which administers to the patient a therapeutic protein in the form of an active protein that is deficient or deficient in the disease or disorder state. However, current therapies present challenges, including frequent treatment, development of an immune response to the therapeutic protein, and difficulty in targeting the therapeutic protein to the affected tissue, cellular, or subcellular compartments. Gene therapy offers advantages including reduced treatment times and sustained efficacy.
Provided herein are compositions for gene therapy vectors that provide improvements in gene therapy, such as providing more therapeutic protein where needed, thus improving therapeutic efficacy. Such challenges are addressed herein by improving the expression and cellular uptake or delivery of therapeutic proteins and intracellular or subcellular targeting. Specific tools or components provided herein include (but are not limited to): signal peptides for increased secretion (e.g., Binding Immunoglobulin Protein (BiP) and Gaussia signal peptides); and peptides that increase endocytosis of the therapeutic protein (e.g., peptides that bind with high affinity to CI-MPR to increase cellular uptake and lysosomal delivery). Such peptides are fused to therapeutic proteins encoded by gene therapy vectors. In some embodiments, the peptide is an IGF2 (insulin-like growth factor 2) peptide or a variant thereof. In some embodiments, it is contemplated that the gene therapy vectors provided herein comprise a nucleic acid encoding a therapeutic protein fused to a peptide that binds with high affinity and CI-MPR to optimize the efficacy of gene therapy.
Gene therapy constructs designed for use in enzyme replacement gene therapy. Translation initiation sequences include, but are not limited to, Kozak sequences or IRES sequences located at the 5' end of the construct, such as CrPV IRES; followed by a nucleic acid encoding a signal peptide for one or more of the GAA signal peptides; a nucleic acid encoding an antitrypsin inhibitor; and a nucleic acid encoding a BiP sequence. These are followed by nucleic acids encoding a cell targeting domain, which may be vIGF-2, HIRMab or TfRMab or other cell targeting peptides or proteins. The gene therapy construct further comprises a nucleic acid encoding a linker and a nucleic acid encoding a proofreading enzyme or an enzymatically active fragment thereof, wherein the linker connects the cell-targeting domain to the proofreading enzyme or an enzymatically active fragment thereof. Suitable proofreading enzymes include, but are not limited to, alpha-Glucosidase (GAA), alpha-Galactosidase (GLA), Iduronidase (IDUA), iduronate-2-sulfatase (IDS), PPT1, or enzymatically active fragments thereof, and other enzymes found in deficient amounts in an individual.
Intracellular targeting of therapeutic proteins
The N-linked carbohydrates of most lysosomal proteins are modified to contain a specific carbohydrate structure called mannose 6-phosphate (M6P). M6P is a biological signal that enables transport of lysosomal proteins to lysosomes via membrane-bound M6P receptors. Enzyme replacement therapy for lysosomal storage diseases uses the M6P receptor for uptake and delivery of therapeutic proteins to lysosomes. Certain therapies do not utilize the M6P receptor, including
And other recombinant human GCase forms, utilize mannose receptors capable of binding terminal mannose on proteinaceous glycans and delivering to lysosomes. Some enzyme replacement therapies face problems: small amounts of M6P were present on the enzyme therapeutics, which required higher doses to achieve therapeutic efficacy. This results in substantially longer infusion times, higher probability of generating an immune response to the therapeutic agent, and higher drug requirements, requiring increased protein manufacture, resulting in increased costs.
CI-MPR captures the M6P-containing lysosomal enzyme from the circulation. The receptor has different binding domains for M6P and insulin-like growth factor (domains 1-3 and 7-9, see fig. 2), and is therefore also known as IGF 2/mannose-6-phosphate receptor or IGF 2/CI-MPR. Such receptors are useful for enzyme replacement therapy targeting variants containing M6P or IGF2 or IGF 2. The binding affinities of this receptor for these ligands (including insulin-like growth factor) are provided in table 1. Notably, IGF2 peptide has higher binding affinity for CI-MPR than mono-or di-phosphorylated oligosaccharides.
Therapeutic fusion proteins for gene therapy
Provided herein are therapeutic fusion proteins produced from gene therapy vectors. In some embodiments, the fusion protein is secreted by a cell transduced with a gene therapy vector encoding the fusion protein. In some embodiments, the transduced cell is within a tissue or organ (e.g., liver). Once secreted from the cell, the fusion protein is transported through the vascular system of the patient and reaches the relevant tissue. In some embodiments, the therapeutic fusion protein is engineered to have improved secretion. In some embodiments, the fusion protein comprises a signal peptide for increasing secretion levels compared to a corresponding therapeutic protein or a fusion protein comprising a therapeutic protein but lacking the signal peptide.
In some embodiments, the provided gene therapy vectors are engineered to address the problems associated with gene therapy with respect to delivery of therapeutic proteins. For example, in some cases, if an insufficient amount of a therapeutic protein is delivered into a cell in need of the therapeutic protein, gene therapy may not achieve the intended treatment simply by producing a sufficient amount of the therapeutic protein in the patient due to, for example, physical and/or biological barriers that hinder the distribution of the therapeutic protein to the desired site. Thus, even if gene therapy is able to submerge blood or tissue to a saturation point with high concentrations of therapeutic proteins, gene therapy may not be sufficiently therapeutic. In addition, non-productive clearance pathways may remove the vast majority of therapeutic proteins. Even if a therapeutic protein is transported from the vascular system to an interstitial space within a tissue (e.g., a muscle fiber), a sufficient therapeutic effect cannot be ensured. In order to effectively treat lysosomal storage disorders, a therapeutically effective amount of the therapeutic protein must be endocytosed and lysosomal delivered to produce meaningful efficacy. The present disclosure addresses these problems by providing gene therapy vectors encoding fusion proteins comprising peptides capable of endocytosing a therapeutic protein into a therapeutic target cell to produce an effective therapy. In some embodiments, the peptide capable of endocytosis is a CI-MPR binding peptide. In some embodiments, the CI-MPR binding peptide is a vIGF2 peptide.
Provided herein are gene therapy vectors encoding fusion proteins comprising peptides capable of endocytosing a therapeutic protein into a therapeutic target cell. In some embodiments, the gene therapy vector encodes a fusion protein comprising a therapeutic protein and a peptide that binds CI-MPR. These fusion proteins, when expressed from a gene therapy vector, target a therapeutic protein (e.g., an enzyme replacement therapeutic agent) to a cell in need thereof, increase delivery into or cellular uptake by these cells, and target the therapeutic protein to a subcellular location (e.g., a lysosome). In some embodiments, the peptide is an IGF2 peptide or variant thereof, which can target the therapeutic protein to a lysosome. Furthermore, in some embodiments, the fusion proteins herein further comprise a signal peptide that increases secretion, such as a BiP signal peptide or a Gaussia signal peptide. In some embodiments, the fusion protein comprises a linker sequence. In some embodiments, a nucleic acid encoding a fusion protein herein comprises an internal ribosomal entry sequence.
Provided herein are therapeutic proteins for gene therapy comprising a vIGF2 peptide. Exemplary proteins are provided in table 2 below.
The components of the fusion proteins provided herein are further described below.
Peptides that bind CI-MPR (e.g., vIGF2 peptide)
Provided herein are peptides that bind CI-MPR. Fusion proteins comprising these peptides and therapeutic proteins, when expressed from a gene therapy vector, target the therapeutic protein to cells in need thereof, increase cellular uptake of these cells, and target the therapeutic protein to subcellular locations (e.g., lysosomes). In some embodiments, the peptide is fused to the N-terminus of the therapeutic peptide. In some embodiments, the peptide is fused to the C-terminus of the therapeutic protein. In some embodiments, the peptide is a vIGF2 peptide. Some vIGF2 peptides maintained high affinity binding to CI-MPR, while their affinity for IGF1 receptor, insulin receptor, and IGF binding protein (IGFBP) was reduced or eliminated. Thus, some variant IGF2 peptides are substantially more selective and have reduced safety risks as compared to wt IGF 2. The vIGF2 peptides herein include those having the amino acid sequence of SEQ ID No. 31. Variant IGF2 peptides also include those having variant amino acids at positions 6, 26, 27, 43, 48, 49, 50, 54, 55, or 65 as compared to wt IGF2(SEQ ID NO: 1). In some embodiments, the vIGF2 peptide has a sequence with one or more substitutions from the group consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, a54R, L55R, and K65R. In some embodiments, the vIGF2 peptide has a sequence with an E6R substitution. In some embodiments, the vIGF2 peptide has a sequence with an F26S substitution. In some embodiments, the vIGF2 peptide has a sequence with Y27L substitutions. In some embodiments, the vIGF2 peptide has a sequence with a V43L substitution. In some embodiments, the vIGF2 peptide has a sequence with an F48T substitution. In some embodiments, the vIGF2 peptide has a sequence with a R49S substitution. In some embodiments, the vIGF2 peptide has a sequence with an S50I substitution. In some embodiments, the vIGF2 peptide has a sequence with an a54R substitution. In some embodiments, the vIGF2 peptide has a sequence with L55R substitutions. In some embodiments, the vIGF2 peptide has a sequence with a K65R substitution. In some embodiments, vIGF2 peptide has a sequence with E6R, F26S, Y27L, V43L, F48T, R49S, S50I, a54R, and L55R substitutions. In some embodiments, the vIGF2 peptide has an N-terminal deletion. In some embodiments, the vIGF2 peptide has an N-terminal deletion of one amino acid. In some embodiments, the vIGF2 peptide has an N-terminal deletion of two amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of three amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of four amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of four amino acids and substitutions of E6R, Y27L, and K65R. In some embodiments, the vIGF2 peptide has an N-terminal deletion of four amino acids and substitutions of E6R and Y27L. In some embodiments, the vIGF2 peptide has an N-terminal deletion of five amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of six amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of seven amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of seven amino acids and substitutions of Y27L and K65R.
Internal ribosome entry sequence
Provided herein are gene therapy constructs useful for treating disorders, further comprising an Internal Ribosome Entry Sequence (IRES) for increasing gene expression by bypassing the bottleneck of translation initiation. Suitable internal ribosome entry sequences for optimizing gene therapy expression include, but are not limited to, cricket paralysis virus (CrPV) IRES, picornavirus IRES, foot and mouth disease virus IRES, kaposi's sarcoma-associated herpesvirus IRES, hepatitis a IRES, hepatitis c IRES, pestivirus IRES, cricket paralysis virus IRES, gahnia virus IRES, merremik disease virus IRES, and other suitable IRES sequences. In some embodiments, the gene therapy construct comprises a CrPV IRES. In some embodiments, the CrPV IRES has the following nucleic acid sequence: AAAAATGTGATCTTGCTTGTAAATACAATTTTGAGAGGTTAATAAATTACAAGTAGTGCTATTTTTGTATTTAGGTTAGCTATTTAGCTTTACGTTCCAGGATGCCTAGTGGCAGCCCCACAATATCCAGGAAGCCCTCTCTGCGGTTTTTCAGATTAGGTAGTCGAAAAACCTAAGAAATTTACCTGCT (SEQ ID NO: 12). In some embodiments, the CrPV IRES sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID No. 12.
Signal peptide
In some embodiments, the gene therapy constructs provided herein further comprise a signal peptide that improves secretion of the therapeutic protein from a cell transduced with the gene therapy construct. In some embodiments, the signal peptide improves protein processing of the therapeutic protein and facilitates translocation of nascent polypeptide-ribosome complexes to the ER and ensures proper co-and post-translational modification. In some embodiments, the signal peptide is located (i) upstream of the signal translation initiation sequence, (ii) between the translation initiation sequence and the therapeutic protein, or (iii) downstream of the therapeutic protein. Signal peptides useful in gene therapy constructs include, but are not limited to, immunoglobulin protein Binding (BiP) signal peptides and Gaussia signal peptides from the HSP70 protein family (e.g., HSPA5, heat shock protein family a member 5), and variants thereof. These signal peptides have an ultra-high affinity for signal recognition particles. Examples of BiP and Gaussia amino acid sequences are provided in table 5 below. In some embodiments, the signal peptide has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID Nos. 13-17. In some embodiments, the signal peptide differs from a sequence selected from the group consisting of SEQ ID Nos. 13-17 by 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid.
BiP signal peptide-Signal Recognition Particle (SRP) interactions facilitate translocation to the ER. This interaction is illustrated in fig. 20.
The Gaussia signal peptide is derived from the marine copepod (Gaussia princeps) luciferase and directs increased protein synthesis and secretion of therapeutic proteins fused to this signal peptide. In some embodiments, the Gaussia signal peptide has an amino acid sequence having at least 90% identity to SEQ ID NO: 32. In some embodiments, the signal peptide differs from SEQ ID NO:32 by 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid.
Joint
In some embodiments, the gene therapy constructs provided herein comprise a linker between the targeting peptide and the therapeutic protein. In some embodiments, these linkers maintain proper spacing and reduce steric clash between the vIGF2 peptide and the therapeutic protein. In some embodiments, the linker comprises repeating glycine residues, repeating glycine-serine residues, and combinations thereof. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids. Suitable linkers for the gene therapy constructs herein include, but are not limited to, those provided in table 6 below.
Translation initiation sequence
Gene therapy constructs provided herein comprise a vector having a translation initiation sequence (e.g., a Kozak sequence, which facilitatesIn initiating translation of the mRNA). The Kozak sequences encompassed herein have the common sequence of (gcc) RccATGG (SEQ ID NO:27), with lower case letters indicating the most common base at that position and base changes, and upper case letters indicating highly conserved bases with only minor changes. R indicates a purine (adenine or guanine) consistently observed at that position. The sequence in parentheses (gcc) has no significance. In some embodiments, the Kozak sequence comprises the sequence AX1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of which is any nucleotide. In some embodiments, X1Contains A. In some embodiments, X2Contains G. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence differs from the sequence of AAGATGA (SEQ ID NO:29) by one or two nucleotides. In some embodiments, the Kozak sequence provided herein has the sequence of AAGATGA (SEQ ID NO: 29). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence differs from the sequence of GCAAGATG (SEQ ID NO:44) by one or two nucleotides. In some embodiments, the Kozak sequence comprises GCAAGATG (SEQ ID NO: 44). In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 85% identity to CACCATG (SEQ ID NO: 47). In some embodiments, the Kozak sequence differs from the sequence of CACCATG (SEQ ID NO:47) by one or two nucleotides. In some embodiments, the Kozak sequence comprises CACCATG (SEQ ID NO: 47).
Therapeutic proteins
Gene therapy constructs provided herein comprise nucleic acids encoding therapeutic proteins for treating a genetic disorder resulting from the absence of a protein or a defective protein due to a genetic defect in an individual. The therapeutic protein expressed from the gene therapy construct replaces the absent protein or the defective protein. Thus, a therapeutic protein is selected based on the genetic defect in an individual in need of treatment. In some embodiments, the therapeutic protein is a structural protein. In some embodiments, the therapeutic protein is an enzyme. In some embodiments, the therapeutic protein is a regulatory protein. In some embodiments, the therapeutic protein is a receptor. In some embodiments, the therapeutic protein is a peptide hormone. In some embodiments, the therapeutic protein is a cytokine or chemokine.
In some embodiments, the gene therapy constructs herein can encode an enzyme, for example an enzyme having a genetic defect in an individual having a lysosomal storage disorder. In some embodiments, the gene therapy construct encodes a lysosomal enzyme, such as a glycosidase, protease, or sulfatase. In some embodiments, the enzymes encoded by the gene therapy constructs provided herein include (but are not limited to): alpha-D-mannosidase; n-aspartyl- β -glucosaminidase; beta-galactosidase; a ceramidase; a fucosidase; galactocerebrosidase; arylsulfatase A; n-acetylglucosamine-1-phosphotransferase; iduronate sulfatase; n-acetylglucosaminidase; acetyl-CoA alpha-glucosaminide acetyltransferase; n-acetylglucosamine 6-sulfatase; beta-glucuronidase; hyaluronic acid enzyme; a sialidase; a sulfatase; sphingomyelinase; acid beta-mannosidase; cathepsin K; 3-hexosidase a; beta-hexosidase B; alpha-N-acetylgalactosaminidase; sialic acid transporter protein (sialin); hexosidase A; a beta-glucosidase; alpha-iduronidase; alpha-galactosidase a; β -glucocerebrosidase; a lysosomal acid lipase; glycosaminoglycan alpha-L-iduronic aldehyde hydrolase; iduronate-2-sulfatase; n-acetylgalactosamine-6-sulfatase; glycosaminoglycan N-acetylgalactosamine 4-sulfatase; an alpha-glucosidase; heparan sulphamidase; gp-91 subunit of NADPH oxidase; adenosine deaminase; cyclin-dependent kinase-like 5; and palmitoyl protein thioesterase 1. In some embodiments, the enzyme encoded by the gene therapy constructs provided herein comprises an alpha-glucosidase. In some embodiments, the therapeutic protein is associated with a genetic disorder selected from the group consisting of: CDKL5 deficiency, cystic fibrosis, alpha-thalassemia and beta-thalassemia, sickle cell anemia, marfan's syndrome, fragile X syndrome, huntington's disease, hemochromatosis, congenital deafness (non-syndromic), tay-saxose disease, familial hypercholesterolemia, duchenne muscular dystrophy, stedt's disease, ewing's syndrome, choroideremia, achromatopsia, X-linked retinoschisis, hemophilia, wir-austenitic syndrome, X-linked chronic granulomatosis, aromatic L-amino acid decarboxylase deficiency, recessive dystrophic epidermolysis bullosa, alpha 1 antitrypsin deficiency, hakinson-gilford progeria syndrome (HGPS), noonan syndrome, X-linked severe combined immunodeficiency syndrome (X-SCID). In some embodiments, the therapeutic protein is selected from the group consisting of: CDKL5, connexin 26, hexosidase A, LDL receptor, dystrophin, CFTR, β -globin, HFE, huntingtin, ABCA4, myosin VIIA (MYO7A), Rab convoyin-1 (REP1), cyclic nucleotide-gated channel β 3(CNGB3), retinoschisin 1(RS1), heme subunit β (HBB), factor IX, WAS, cytochrome B-245 β chain, Dopa Decarboxylase (DDC), collagen type VII α 1 chain (COL7a1), serine protease inhibitor family a member 1(SERPINA1), LMNA, PTPN11, SOS1, RAF1, KRAS, and IL2 receptor γ genes.
Examples of Gene therapy vectors
Gene therapy vectors and compositions
Provided herein are gene therapy vectors in which a nucleic acid (e.g., DNA) encoding a therapeutic fusion protein (e.g., a vIGF2 fusion) optionally has a signal peptide. The gene therapy vector optionally comprises an internal ribosome entry sequence. Retroviral (e.g., lentiviral) derived vectors are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of a transgene and its propagation in daughter cells. Lentivirus and adeno-associated virus vectors have the additional advantage over vectors derived from oncogenic retroviruses, such as murine leukemia virus, in that they are capable of transducing non-proliferating cells, such as hepatocytes and neurons. They also have the additional advantage of low immunogenicity.
Exemplary gene therapy vectors herein encode therapeutic proteins and therapeutic fusion proteins comprising a vIGF2 peptide. Nucleic acids encoding exemplary fusion protein amino acid sequences are provided in table 7 below.
In some embodiments, the vectors provided herein comprising a nucleic acid encoding a desired therapeutic fusion protein (e.g., a vIGF2 fusion or a signal peptide fusion), optionally with an internal ribosome entry sequence, are adeno-associated viral vectors (a 5/35).
In some embodiments, nucleic acids encoding therapeutic fusion proteins (e.g., vIGF2 fusions) and optionally having internal ribosome entry sequences are cloned into various types of vectors. For example, in some embodiments, the nucleic acid is cloned into a vector, including (but not limited to) plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
Furthermore, in some embodiments, an expression vector encoding a desired therapeutic fusion protein (e.g., a vIGF2 fusion or a signal peptide fusion) and optionally having an internal ribosome entry sequence is provided to a cell in the form of a viral vector. Viral vector technology is described, for example, in Sambrook et al, 2012, Molecular Cloning: A laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY) and other handbooks for virology and Molecular biology. Viruses suitable for use as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, a suitable restriction endonuclease site, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
Also provided herein are compositions and systems for gene transfer. A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a suitable platform for gene delivery systems. In some embodiments, the selected gene is inserted into a vector and packaged in a retroviral particle using a suitable technique. The recombinant virus is then isolated and delivered to cells of the subject in vivo or ex vivo. A variety of retroviral systems are suitable for gene therapy. In some embodiments, an adenoviral vector is used. A variety of adenoviral vectors are suitable for gene therapy. In some embodiments, an adeno-associated viral vector is used. A variety of adeno-associated viruses are suitable for gene therapy. In one embodiment, a lentiviral vector is used.
Gene therapy constructs provided herein comprise a vector (or gene therapy expression vector) into which the relevant gene is cloned or which comprises the relevant gene in a manner such that the nucleotide sequence of the vector allows expression (constitutive expression or regulated in some way) of the relevant gene. Vector constructs provided herein include any suitable gene expression vector capable of delivery to the relevant tissue and which will provide for expression of the relevant gene in the selected relevant tissue.
In some embodiments, the vector is an adeno-associated virus (AAV) vector due to the ability of the AAV vector to cross the blood brain barrier and transduce neuronal tissue. In the methods provided herein, the use of any serotype of AAV is contemplated. In certain embodiments, the viral vector of the serotype used is selected from the group consisting of: AAV1 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector, AAV8 vector, AAV9 vector, AAVrhS vector, AAVrh10 vector, AAVrh33 vector, AAVrh34 vector, AAVrh74 vector, AAV Anc80 vector, aavphp.b vector, AAVhu68 vector, AAV-DJ vector and other vectors suitable for gene therapy.
AAV vectors are DNA parvoviruses that are non-pathogenic to mammals. Briefly, AAV-based vectors remove the rep and cap viral genes that represent 96% of the viral genome, leaving two flanking 145 base pair Inverted Terminal Repeats (ITRs) for initiating viral DNA replication, packaging and integration.
Other embodiments include the use of other serotype capsids to produce: AAV1 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector, AAV8 vector, AAV9 vector, AAVrhS vector, AAVrh10 vector, AAVrh33 vector, AAVrh34 vector, AAVrh74 vector, AAV Anc80 vector, aavphp.b vector, AAV-DJ vector and other vectors suitable for gene therapy. Optionally, the AAV viral capsid is AAV2/9, AAV9, AAVrhS, AAVr h10, AAVAnc80, or AAV php.b.
Additional promoter elements (e.g., enhancers) regulate the transcription initiation frequency. Typically, these are located in the region 30-110bp upstream of the start site, although many promoters have been shown to contain functional elements also downstream of the start site. The spacing between promoter elements is typically flexible such that promoter function is retained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements is typically increased to 50bp apart before activity begins to decline. Depending on the promoter, the individual elements appear to function cooperatively or independently to activate transcription.
An example of a promoter capable of expressing a therapeutic fusion protein (e.g., a vIGF2 fusion or a signal peptide fusion) and optionally having an internal ribosome entry sequence, a transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of the aminoacyl tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression and has been shown to be effective in driving expression of transgenes from cloning into lentiviral vectors (see, e.g., Milone et al, mol. Ther.17(8):1453-1464 (2009)). Another example of a promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. Such promoter sequences are strong constitutive promoter sequences capable of driving high levels of expression of any polynucleotide sequence to which they are operably linked. However, other constitutive promoter sequences may sometimes be used, including, but not limited to, the chicken β actin promoter, the P546 promoter, the simian virus 40(SV40) early promoter, the Mouse Mammary Tumor Virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus (Epstein-Barr virus) immediate early promoter, the Rous sarcoma (Rous sarcoma) virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1 a promoter, the heme promoter, and the creatine kinase promoter. Furthermore, it is contemplated that gene therapy vectors are not limited to the use of constitutive promoters. Inducible promoters are also contemplated herein. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline regulated promoters.
To assess the expression of a therapeutic fusion protein (e.g., a vIGF fusion or a signal peptide fusion) optionally having an internal ribosome entry sequence or portion thereof, the expression vector to be introduced into a cell typically contains a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from a population of cells that are intended to be transfected or infected by a viral vector. In other aspects, the selectable marker is typically carried on a separate DNA segment and used in a co-transfection procedure. Selectable markers and reporter genes are sometimes flanked by appropriate regulatory sequences to achieve expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.
Methods and compositions for introduction into cells and expression of genes are suitable for the methods herein. In the case of expression vectors, the vectors are readily introduced into host cells (e.g., mammalian, bacterial, yeast, or insect cells) by any method known in the art. For example, the expression vector is transferred into a host cell by physical, chemical or biological means.
Physical methods and compositions for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene guns, electroporation, and the like. Methods for making cells comprising the vector and/or the exogenous nucleic acid are suitable for the methods herein (see, e.g., Sambrook et al, 2012, Molecular Cloning: A Laboratory Manual, Vol. 1-4, Cold Spring Harbor Press, NY). One method of introducing polynucleotides into host cells is calcium phosphate transfection.
Chemical means and compositions for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, nucleic acid-lipid particles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle). Other methods of targeted delivery of nucleic acids in currently advanced technologies are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable submicron-sized delivery systems.
Where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations for introducing nucleic acids into host cells (in vitro, ex vivo or in vivo) is contemplated. In another aspect, the nucleic acid is associated with a lipid. In some embodiments, the nucleic acid associated with a lipid is encapsulated within the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, linked to the liposome via a linker molecule associated with the liposome and an oligonucleotide, encapsulated in the liposome, complexed with the liposome, dispersed in a solution containing the lipid, mixed with the lipid, combined with the lipid, contained in suspension in the lipid, contained in or complexed with a micelle, or otherwise associated with the lipid. The composition associated with the lipid, lipid/DNA or lipid/expression vector is not limited to any particular structure in solution. For example, in some embodiments, they are present in a bilayer structure, in the form of micelles or with a "collapsed" structure. Alternatively, they may simply be interpenetrated in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which in some embodiments are naturally occurring or synthetic lipids. For example, lipids include droplets of fat naturally occurring in the cytoplasm as well as classes of compounds containing long chain aliphatic hydrocarbons and their derivatives (e.g., fatty acids, alcohols, amines, amino alcohols, and aldehydes).
Suitable lipids are available from commercial sources. For example, in some embodiments, dimyristylphosphatidylcholine ("DMPC") is obtained from Sigma, st.louis, mo.; in some embodiments, dicetyl phosphate ("DCP") is obtained from K & K Laboratories (Plainview, n.y.); in some embodiments, cholesterol ("Choi") is obtained from Calbiochem-Behring; dimyristylphosphatidylglycerol ("DMPG") and other Lipids are commonly available from Avanti Polar Lipids, Inc. Stock solutions of lipids in chloroform or chloroform/methanol are typically stored at about-20 ℃. Chloroform was used as the only solvent because it evaporates more readily than methanol. "liposomes" is a generic term that encompasses a variety of mono-and multilamellar lipid vehicles formed by the production of encapsulated lipid bilayers or aggregates. Liposomes are generally characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components rearrange themselves before forming a closed structure and trap water and dissolved solutes between lipid bilayers (Ghosh et al, 1991Glycobiology 5: 505-10). However, compositions having a structure in solution that is different from the normal vesicle structure are also contemplated. For example, in some embodiments, the lipids present a micellar structure or merely exist as non-uniform aggregates of lipid molecules. Lipid-staining amine-nucleic acid complexes are also contemplated.
Regardless of the method used to introduce the exogenous nucleic acid into the host cell or expose the cell to the therapeutic fusion protein provided herein (e.g., a vIGF2 fusion or a signal peptide fusion), optionally with an internal ribosome entry sequence, various assays are contemplated for confirming the presence of recombinant DNA sequences in the host cell. Such assays include, for example, "molecular biology" assays suitable for the methods herein, such as Southern and Northern blots, RT-PCR and PCR; "biochemical" assays, e.g., detecting the presence or absence of a particular peptide, identify reagents falling within the scope hereof, e.g., by immunological means (ELISA and western blot) or by assays described herein.
The disclosure also provides a vector comprising a therapeutic fusion protein (e.g., a vIGF2 fusion or a signal peptide fusion), optionally with an internal ribosome entry sequence, encoding a nucleic acid molecule. In one aspect, the therapeutic fusion protein vector is capable of being directly transduced into a cell. In one aspect, the vector is a cloning or expression vector, such as, but not limited to, vectors including: one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, microcarriers, double minichromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the vIGF 2-therapeutic fusion protein construct in a mammalian cell. In one aspect, the mammalian cell is a human cell.
Use and method of treatment
Also provided herein are methods of treating a genetic disorder using gene therapy, comprising administering to an individual a nucleic acid encoding a therapeutic fusion protein disclosed herein (e.g., a vIGF2 fusion or a signal peptide-vIGF 2 fusion) and optionally having an internal ribosome entry sequence. Genetic disorders suitable for treatment using the methods herein include disorders in an individual caused by one or more mutations in the genome that result in the absence of an expressed protein or the expression of a dysfunctional protein by a mutant gene.
Also provided herein are pharmaceutical compositions comprising a gene therapy vector, e.g., a gene therapy vector comprising a nucleic acid encoding a therapeutic fusion protein disclosed herein (e.g., a vIGF2 fusion or a signal peptide-vIGF 2 fusion) and optionally having an internal ribosome entry sequence, and a pharmaceutically acceptable carrier or excipient, for use in the preparation of a medicament for the treatment of a genetic disorder.
Genetic disorders suitable for treatment by the methods herein include (but are not limited to): achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid Syndrome, autosomal dominant polycystic kidney Disease, Chack-Marie-Dus Disease (Charcot-Marie-Tooth), colon cancer, Cat's Syndrome (Cri du chat), Crohn's Disease, cystic fibrosis, Derch's Disease, Dunne Syndrome (Duane Syndrome), Duchenne muscular dystrophy, Factor V Laden Disease (Factor V Leiden Thrombophilia), familial hypercholesterolemia, familial Mediterranean fever, Fragile X Syndrome, gaucher's Disease, hemochromatosis, hemophilia, forebrain non-cracking malformation, Huntington's Disease (Huntington ' Disease), Klinefelter Syndrome (Klefelder Syndrome), Marek's Syndrome, muscular dystrophia, neurofibroma, neurofibromyalgia Syndrome, osteogenesis, Parkinson's Disease (Parkinson's Disease), Phenylketonuria, Poland Anomaly, porphyria, progeria, retinitis pigmentosa, Severe Combined Immunodeficiency (SCID), sickle cell Disease, spinal muscular atrophy, Thai-saxophone Disease, thalassemia, trimethylaminouria, Turner Syndrome, jaw face Syndrome, WAGR Syndrome, or Wilson Disease. In some embodiments, the genetic disorder is selected from the group consisting of: CDKL5 deficiency, cystic fibrosis, alpha-thalassemia and beta-thalassemia, sickle cell anemia, marfan's syndrome, fragile X syndrome, huntington's disease, hemochromatosis, congenital deafness (non-syndromic), tay-saxose disease, familial hypercholesterolemia, duchenne muscular dystrophy, stedt's disease, ewing's syndrome, choroideremia, achromatopsia, X-linked retinoschisis, hemophilia, wir-austenitic syndrome, X-linked chronic granulomatosis, aromatic L-amino acid decarboxylase deficiency, recessive dystrophic epidermolysis bullosa, alpha 1 antitrypsin deficiency, hakinson-gilford progeria syndrome (HGPS), noonan syndrome, X-linked severe combined immunodeficiency syndrome (X-SCID).
In some embodiments, the genetic disorder suitable for treatment using the methods provided herein is a lysosomal storage disorder. In some embodiments, gene therapy is used herein to deliver a deletion or defective enzyme to a patient to treat a lysosomal storage disorder. In some embodiments, the methods herein deliver an enzyme fused to vIGF2 or to a signal peptide to a patient in order to deliver the enzyme into a cell in need thereof. In some embodiments, the lysosomal storage disease is selected from the group consisting of: aspartylglucamine diabetes, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, MuldoHoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hewler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Moquini disease type A, Moquine disease type B, Maruto-Lami disease, Sprius disease, Niemann-pick disease type A, Niemann-pick disease type B, Niemann-pick disease type C1, Niemann-pick disease type C2, Ocimir disease type I and Ocimier disease type II. In some embodiments, the lysosomal storage disease is selected from the group consisting of: deficiency of activating factor, GM 2-gangliosidosis; GM 2-gangliosidosis, AB variant; alpha-mannosidosis (type 2, moderate form; type 3, neonatal, severe); beta-mannoside storage disease; aspartylglucosaminuria; lysosomal acid lipase deficiency; cystinosis (type of late onset juvenile or juvenile nephropathy; nephropathic nature in infants); christian-dofmann syndrome; neutral lipid storage disease with myopathy; NLSDM; danong disease (Danon disease); fabry disease; late onset fabry disease type II; fabry disease; farber fatty granulomatosis; fucoside storage disorders; galactosialistorage disorder (combined neuraminidase and beta-galactosidase deficiency); gaucher disease; gaucher disease type II; gaucher disease type III; gaucher disease type IIIC; atypical gaucher disease due to sphingolipid activator protein C deficiency; GM 1-gangliosidosis (advanced infantile/juvenile GM 1-gangliosidosis; adult/chronic GM 1-gangliosidosis); globulocyte leukodystrophy, krabbe's disease (late onset infant type; juvenile onset type; adult onset type); atypical krabbe's disease due to sphingolipid activator protein a deficiency; metachromatic leukodystrophy (juvenile; adult); partial sulfaterebroside deficiency; pseudoarylsulfatase a deficiency; metachromatic leukodystrophy due to sphingolipid activator B deficiency; mucopolysaccharide storage disorder: MPS I, heler syndrome; MPS I, heler-schey syndrome; MPS I, schey syndrome; MPS II, hunter syndrome; MPS II, hunter syndrome; sanfilippo syndrome/MPS IIIA type a; sanfilippo syndrome/MPS IIIB type B; sanfilippo syndrome/MPS IIIC type C; sanfilippo syndrome/MPS IIID type D; morquio syndrome, type a/MPS IVA; morquio syndrome, type B/MPS IVB; MPS IX hyaluronidase deficiency; MPS VI marlotte-lamide syndrome; MPS VII sri syndrome; salivary gland disease type I, II mucolipidosis; i-cell disease, Leluo disease, mucolipidosis II; shampooing malnutrition/mucolipidosis type III; mucolipidosis IIIC/ML III GAMMA; type IV mucolipidosis; polythiol esterase deficiency; niemann-pick disease (type B; type C1/chronic neurological form; type C2; type D/Nova scotia); neuronal ceroid lipofuscinosis: CLN6 disease, atypical late infant, late onset variant, early childhood; pasteur/juvenile NCL/CLN3 disease; finnish variant late infant CLN 5; jenski-biziksky disease/late infantile CLN2/TPP1 disease; kuves/adult onset NCL/CLN4 disease (type B); northern epilepsy/variant late infant CLN 8; Santaiwaii-Hardiya/infant CLN1/PPT disease; pompe disease (glycogen storage disease type II); late onset pompe disease; dense bone developmental disorder; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sandhoff disease/GM 2 gangliosidosis; sinderler disease (type III/moderate, variable); qi disease of the Shen; sara disease; infantile asialostorage disease (ISSD); spinal muscular atrophy with progressive myospasm epilepsy (SMAPME); Tay-saxophone/GM 2 gangliosidosis; juvenile onset tay-saxophone disease; late onset tay-saxophone; klissinostian syndrome; lowboy eye-brain-kidney syndrome; type 4J chak-mary-dus disease, CMT 4J; ulis-von syndrome; bilateral temporal occipital multicephalic gyrus (BTOP); x-linked hypercalcemic nephrolithiasis, dent-1; and dengue 2. In some embodiments, the therapeutic protein is associated with a lysosomal storage disease, and the therapeutic protein is selected from the group consisting of: GM 2-activator protein; alpha-mannosidase; MAN2B 1; lysosomal β -mannosidase; a glycosylasparaginase enzyme; a lysosomal acid lipase; cystine transporter protein (cystinosin); a CTNS; PNPLA 2; lysosomal associated membrane protein-2; alpha-galactosidase a; GLA; acid ceramidase; an alpha-L-fucosidase; protective protein/cathepsin a; acid beta-glucosidase; GBA; a PSAP; beta-galactosidase-1; GLB 1; galactosylceramide beta-galactosidase; GALC; a PSAP; arylsulfatase A; ARSA; alpha-L-iduronidase; iduronate 2-sulfatase; heparan N-sulfatase; n- α -acetylglucosaminidase; heparan acetyl-CoA alpha-glucosaminide acetyltransferase; n-acetylglucosamine 6-sulfatase; galactosamine-6-sulfatase; beta-galactosidase; hyaluronic acid enzyme; arylsulfatase B; beta-glucuronidase; neuraminidase; NEU 1; gamma subunits of N-acetylglucosamine-1-phosphotransferase; mucrolipin-1; sulfatase modification factor-1; acid sphingomyelinase; SMPD 1; NPC 1; and NPC 2.
In some embodiments, a gene encoding a therapeutic protein is delivered into a cell in need of the therapeutic protein via treatment by the methods herein. In some embodiments, the treatment delivers the gene to all somatic cells in the individual. In some embodiments, the therapeutic replacement targets a defective gene in the cell. In some embodiments, the cells treated ex vivo to express the therapeutic protein are delivered to an individual.
Gene therapy for the conditions disclosed herein provides therapeutic results that are superior to conventional therapies, including enzyme replacement therapy, because it does not require long-term infusion therapy. In addition, it reduces the risk of an individual developing an immune response to the therapeutic protein, which is often experienced in individuals receiving enzyme replacement therapy.
Definition of
As used herein, "ex vivo gene therapy" refers to a method in which patient cells are genetically modified outside of a subject, for example, to express a therapeutic gene. The cells with the new genetic information are then returned to the subject from which they were derived.
As used herein, "in vivo gene therapy" refers to a method in which a vector carrying a therapeutic gene is directly administered to a subject.
As used herein, "fusion protein" and "therapeutic fusion protein" are used interchangeably herein and refer to a therapeutic protein having at least one additional protein, peptide, or polypeptide attached thereto. In some cases, a fusion protein is a single protein molecule containing two or more proteins, or fragments thereof, covalently linked via peptide bonds within their respective peptide chains without chemical linkers. In some embodiments, the fusion protein comprises a therapeutic protein and a signal peptide (a peptide that increases endocytosis of the fusion protein) or both. In some embodiments, the peptide that increases endocytosis is a CI-MPR binding peptide.
As used herein, "vector" or "gene therapy vector" used interchangeably herein refers to a gene therapy delivery vehicle or vector that delivers a therapeutic gene to a cell. A gene therapy vector is any vector suitable for use in gene therapy, such as any vector suitable for the therapeutic delivery of a nucleic acid polymer (encoding a polypeptide or variant thereof) into a target cell (e.g., a sensory neuron) of a patient. In some embodiments, the gene therapy vector delivers a nucleic acid encoding a therapeutic protein or therapeutic fusion protein to a cell that expresses the therapeutic protein or fusion and is secreted from the cell. The vector may be of any type, for example it may be a plasmid vector or a minicircle DNA. Typically, the vector is a viral vector. These include both gene-disabled viral (e.g., adenoviral) vectors and non-viral vectors (e.g., liposomes). The viral vector may, for example, be derived from an adeno-associated virus (AAV), a retrovirus, lentivirus, herpes simplex virus or adenovirus. Vectors of AAV origin. The vector may comprise an AAV genome or derivative thereof.
As used herein, "construct" refers to a nucleic acid molecule or sequence that encodes a therapeutic protein or fusion protein and optionally includes additional sequences (e.g., translation initiation sequences or IRES sequences).
As used herein, "plasmid" refers to a circular double-stranded DNA unit that replicates independently of chromosomal DNA within a cell.
As used herein, "promoter" refers to a site on DNA that binds to the enzyme RNA polymerase and initiates transcription of DNA into RNA.
As used herein, "somatic cell therapy" refers to a method in which gene expression in cells that will be corrective for a patient rather than inherited to the next generation is manipulated. Somatic cells include all non-germ cells in the human body.
As used herein, "somatic cell" refers to all cells of the body except germ cells.
As used herein, "tropism" refers to the preference of a vector (e.g., a virus) for a certain cell or tissue type. Various factors determine the ability of the vector to infect a particular cell. Viruses, for example, must bind to specific cell surface receptors to enter cells. If the cell does not express the essential receptor, the virus is generally unable to infect the cell.
The term "transduction" is used to refer to the in vivo or in vitro administration/delivery of a nucleic acid encoding a therapeutic protein to a target cell via a replication-deficient rAAV of the present disclosure, thereby causing the recipient cell to express a functional polypeptide. Transduction of cells with a gene therapy vector (e.g., rAAV) of the present disclosure results in sustained expression of the polypeptide or RNA encoded by the rAAV. Accordingly, the present disclosure provides methods of administering/delivering a gene therapy vector (e.g., rAAV) encoding a therapeutic protein to a subject by intrathecal, intraretinal, intraocular, intravitreal, intracerebroventricular, intraparenchymal, or intravenous routes, or any combination thereof. By "intrathecal" delivery is meant delivery into the subintimal space of the brain or spinal cord. In some embodiments, intrathecal administration is via intracisternal administration.
The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein, and in some cases refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy. "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic animals and farm animals, as well as laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like. In some embodiments, the mammal is a human.
As used herein, in some instances, the terms "treating", "ameliorating", and the like, refer to administering an agent or performing a procedure for the purpose of obtaining a therapeutic effect (including inhibiting, alleviating, reducing, preventing, or altering at least one aspect or marker of a disorder) in a statistically significant manner or in a clinically significant manner. The terms "improving" or "treating" do not indicate or imply a cure for the underlying condition. As used herein, "treating" or "ameliorating" (etc.) may include treating a mammal, particularly a human, and includes: (a) preventing a disorder or symptoms of a disorder from occurring in a subject who may be predisposed to the disorder but has not yet been diagnosed as diseased (e.g., including disorders that may be associated with or caused by a primary disorder); (b) inhibiting the disorder, i.e., arresting its development; (c) relieving the condition, i.e., causing regression of the condition; and (d) ameliorating at least one symptom of the disorder. Treatment may refer to any indicator of success in treating or ameliorating or preventing a disorder, including any objective or subjective parameter, such as elimination; (iii) alleviating; reducing symptoms or making the condition more tolerable to the patient; slowing the rate of degeneration or decline; or make the final point of degradation less debilitating. Treating or ameliorating the symptoms is based on one or more objective or subjective parameters; including the results of the examination by the physician. Thus, the term "treating" includes administering a compound or agent of the invention to prevent or delay, reduce or arrest or inhibit the development of the symptoms or conditions associated with the disorder. The term "therapeutic effect" refers to a reduction, elimination, or prevention of a disorder, a symptom of a disorder, or a side effect of a disorder in a subject.
The term "affinity" refers to the strength of binding between a molecule and its binding partner or receptor.
As used herein, the phrase "high affinity" refers to, for example, a therapeutic fusion containing such a peptide that binds CI-MPR with an affinity to CI-MPR that is about 100 to 1,000-fold or 500 to 1,000-fold that of a therapeutic protein without the peptide. In some embodiments, the affinity is at least 100, at least 500, or at least 1000-fold greater than without the peptide. For example, when a therapeutic protein and CI-MPR are combined at relatively equal concentrations, a peptide with high affinity will bind to the available CI-MPR in order to shift the equilibrium towards a high concentration of the resulting complex.
As used herein, "secreted" refers to the release of a protein from a cell into the blood stream, e.g., to be carried to the relevant tissue or site of action of a therapeutic protein. When the gene therapy product is secreted into the interstitial space of an organ, secretion may allow cross-correction of neighboring cells.
As used herein, "delivery" means drug delivery. In some embodiments, the delivery process means transporting the drug substance (e.g., a therapeutic protein or fusion protein produced from a gene therapy vector) from outside the cell (e.g., blood, tissue, or interstitial space) into the therapeutically active target cell of the drug substance.
As used herein, "engineering" or "protein engineering" refers to the manipulation of the structure of a protein by providing appropriate nucleic acid sequences encoding the protein in order to produce a protein of a desired property or having a particular structure.
In some instances, a "therapeutically effective amount" means an amount sufficient to effect treatment of a disorder when administered to a subject for treating the disorder.
As used herein, the term "about" a number refers to a range that spans from less than 10% of the number to greater than 10% of the number, and includes values within the range (e.g., the number itself).
As used herein, the term "comprising" or "comprises" an element or elements of a claim means that those elements do not preclude the inclusion of one or more additional elements.
Examples
The following examples are given for the purpose of illustrating various embodiments of the invention and are not intended to limit the invention in any way. The examples, together with the methods described herein, are presently representative of the preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Variations thereof and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.
Example 1: binding of variant IGF2 peptides to CI-MPR receptors
Surface Plasmon Resonance (SPR) experiments were performed using Biacore to measure the binding of wild-type and variant IGF2(vIGF2) to CI-MPR receptors. The wild-type human mature IGF2 peptide (wt IGF2) has the sequence shown in SEQ ID NO: 1. The vIGF2 sequence differs from wt IGF2 in that it lacks residues 1-4 and contains the following mutations: E6R, Y27L and K65R. It has the amino sequence: SRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE (SEQ ID NO: 31). vIGF2 also has an N-terminal linker with the sequence GGGGSGGGG (SEQ ID NO: 18). The combined sequence was GGGGSGGGGSRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE (SEQ ID NO: 43). Figure 4 shows that, as expected, wild-type IGF2 peptide binds with high affinity (0.2nM) to CI-MPR receptor. Figure 5 shows that the variant IGF2 peptide (vIGF2) also bound with high affinity (0.5nM) to the CI-MPR receptor. These data indicate that vIGF2 peptide has high affinity for the expected CI-MPR receptor for targeting therapeutic agents to lysosomes.
SPR was used to measure peptides binding to the insulin receptor to assess potential side effects. Insulin binds to the insulin receptor with high affinity (-8 nM; data not shown). Wild-type IGF2 and vIGF2 were tested, wherein vIGF2 has sequence SRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE (SEQ ID NO:31) with an N-terminal linker having the sequence GGGGSGGGG (SEQ ID NO: 18). Fig. 8 shows that wild-type IGF2 also binds insulin receptor with relatively high affinity (-100 nM). The IGF2 peptide from the Biomarin/zytor IGF2-GAA fusion protein (BMN-701) also binds to the insulin receptor with high affinity and was shown to cause hypoglycemia in clinical trials. Figure 9 shows that there was no measurable binding of vIGF2 peptide to insulin receptor. These data indicate that vIGF2 peptide confers a higher safety profile than the wt IGF2 peptide fusion.
The interaction of vIGF2 peptide with IGF1 receptor was characterized using the same SPR binding assay. Fig. 10 shows that wild-type IGF2 peptide binds IGF1 receptor with relatively high affinity (-100 nM). Figure 11 shows no measurable binding of vIGF2 peptide to IGF1 receptor, indicating an improved safety profile compared to wt IGF 2.
Example 2: vIGF2 converts low affinity ligands for CI-MPR to high affinity ERT
A vIGF2 peptide (SEQ ID NO:31) with an N-terminal linker (SEQ ID NO:18) was chemically coupled to the alpha-glucosidase, referred to herein as vIGF 2-alpha-glucosidase to determine whether the vIGF2 peptide could improve affinity for CI-MPR. As shown in figure 6, the binding affinities of the arabinosidase-alpha and vIGF 2-arabinosidase-alpha were directly compared using a CI-MPR plate binding assay in a CI-MPR coated 96-well ELISA plate. Unbound enzyme was washed away before measuring bound enzyme activity. Varying concentrations of both enzyme preparations were used with or without free WT IGF2 peptide. vIGF2 substantially improved the affinity for CI-MPR. Furthermore, binding of vIGF 2-arabinosidase-a was blocked by free WT IGF2, indicating that binding is IGF2 dependent. (data not shown.) coupling of vIGF2 peptide did not impair GAA enzyme activity.
vIGF2 was coupled to recombinant human N-acetyl- α -D-glucosaminidase (rhNAGLU). Rhnaglu, a lysosomal enzyme lacking M6P, was used to determine whether peptides could convert non-ligands to high affinity ligands for CI-MPR. In this experiment, rhNAGLU and vIGF2-rhNAGLU were compared directly using CI-MPR coated plates in conjunction with the assay. Unbound enzyme was washed away before measuring bound enzyme activity. Varying concentrations of both enzyme preparations were used with or without free vIGF2 peptide. As shown in fig. 7, vIGF2-rhNAGLU had significantly higher affinity for CI-MPR than rhNAGLU lacking vIGF 2. Furthermore, vIGF2-rhNAGLU binding was blocked by the free vIGF2 peptide, indicating that receptor binding is specific for the IGF2 peptide. These results indicate that vIGF2 peptide can be used to improve drug targeting to lysosomes.
Example 3: myoblast uptake of vIGF2-GAA fusion protein
vIGF2-GAA fusion protein (same sequence as in example 1-2) was administered and the L6 myoblast uptake of the enzyme was measured. Figure 6 shows better absorption of vIGF2-rhGAA compared to rhGAA and M6P-GAA. Therefore, vIGF2 was effective in targeting GAA to cells.
Example 4: by passingConstructs for ERT for gene therapy delivery
Two different constructs are shown in figure 12. The top panel is a construct encoding "native hGAA" (SEQ ID NO:45) containing a Kozak sequence and a nucleic acid encoding recombinant human GAA with a native signal peptide (SEQ ID NO: 45). The middle panel is the construct Kozak-BiP-vIGF2-2GS-GAA encoding "engineered hGAA" (SEQ ID NO: 23). This construct is characterized by a Kozak sequence, a nucleic acid encoding a BiP signal peptide, a nucleic acid encoding a vIGF2 peptide (having the sequence shown in SEQ ID NO: 31) and a nucleic acid encoding a 2GS linker (SEQ ID NO:18), followed by a nucleic acid encoding recombinant human GAA in which the N-terminal 60 amino acids (SEQ ID NO:46) are removed to prevent premature processing and removal of vIGF 2.
Example 5: enhanced secretion of gene therapy constructs
Engineered hGAA have higher secretion and are capable of interacting with cell surface receptors suitable for cellular uptake and lysosomal targeting
CHO expressing engineered hGAA (described in more detail below) or native hGAA is cultured and conditioned media is collected for measurement of GAA activity. Fig. 15 shows the relative activities of engineered and native hGAA, indicating that the engineered hGAA has increased activity compared to native hGAA, indicating more efficient secretion of the engineered hGAA.
Example 6: analysis of PPT1 in conditioned Medium
Cloning of the PPT1 construct
The PPT1 construct was cloned into pcdna3.1 expression vector (ThermoFisher, catalog # V79020) containing the CMV promoter. The constructs tested included PPT1-1(WT-PPT1) (SEQ ID NO:24), PPT1-2(WT-vIGF2-PPT1) (SEQ ID NO:25), PPT1-29(BiP2aa-vIGF2-PPT1) (SEQ ID NO: 26).
PPT1 secretion and binding
The PPT1 construct was transiently expressed in HEK293T cells for 3 days and PPT1 was secreted into the culture medium. Secreted PPT1 was quantified by western blotting and CI-MPR binding was analyzed using well-established methods. The secreted PPT1 is shown in figure 13. The CI-MPR combination is shown in FIG. 14.
Example 7: testing gene therapy vectors in Pompe disease animal models
Pompe gene therapy: preclinical proof of concept study design
In GAA knockout (GAA KO) mice, preclinical studies were performed using high doses for initial comparison of constructs. The constructs are shown in figure 12. Mice were treated with vehicle or one of the two constructs (native hGAA or engineered hGAA). Mice were administered 5e11 gc/mouse (approximately 2.5e13 gc/kg). GAA knockout mice of 2 months of age were used. Normal (wild type) mice were used as controls. The study design is summarized in figure 16.
Pompe gene therapy: blood plasma
Plasma was collected from wild type (normal) mice or GAA KO mice treated with vehicle or gene therapy vector as indicated, and GAA activity and cell surface binding were measured. The data are summarized in fig. 17, 27 and 19. Similar high GAA levels were seen in mice treated with gene therapy vectors (fig. 17, fig. 18). However, higher cell-targeted receptor binding was observed with the engineered constructs (fig. 19).
Pompe gene therapy: quadriceps muscle
GAA activity and glycogen storage/cytoplasmic vacuolization were assessed in normal (wild-type) and treated GAA KO mice (fig. 28). GAA activity in quadriceps is about 20 times that of the wild type. Glycogen PAS (fig. 29) and immunohistochemistry (fig. 30) were also evaluated. Immunohistochemistry showed that the engineered hGAA had higher lysosomal targeting compared to the wild type. The glycogen reduction of engineered hGAA was more constant according to PAS staining.
Pompe gene therapy: triceps muscle
GAA activity and glycogen storage/cytoplasmic vacuolation were assessed in normal (wild-type) and treated GAA KO mice (fig. 31). The GAA activity is about 10-15 times that of the wild type. Immunohistochemistry and glycogen PAS were also evaluated (fig. 32 and 33). Immunohistochemistry indicated that the engineered hGAA had higher lysosomal targeting compared to wild-type GAA. The glycogen reduction of the engineered hGAA was more constant as measured by PAS staining.
Pompe gene therapy: tibialis Anterior (TA)
GAA activity and glycogen storage/cytoplasmic vacuolation were assessed in normal (wild-type) and treated GAA KO mice (fig. 20). GAA activity in TA is about 15-20 times that of wild type. Immunohistochemistry and glycogen PAS were also evaluated (fig. 21 and 22). Immunohistochemistry indicated that the engineered hGAA had higher lysosomal targeting compared to wild-type GAA. Glycogen levels are close to wild-type levels. The glycogen reduction of engineered hGAA was more constant according to PAS staining.
Pompe gene therapy: brain and spinal cord
GAA activity, glycogen content and glycogen storage/cytoplasmic vacuolization were assessed in normal (wild-type) and treated GAA KO mice (fig. 23). GAA activity in brain is about 5 times lower than that of wild type. Immunohistochemistry and glycogen PAS were also evaluated (fig. 24, 25, 26, 27). Immunohistochemistry indicates that there may be direct transduction of some cells. However, the native construct achieved little to no glycogen clearance. Glycogen levels of the engineered constructs were close to wild-type levels, even though activity was only 20% of wild-type. PAS staining in the spinal cord showed little to no glycogen clearance of the native construct. In the ventral horn, which included motor neurons, engineered constructs were observed to approximate wild-type glycogen levels. Immunohistochemistry indicated direct transduction in spinal cord neurons. Engineered hGAA produced by choroid plexus and neuronal cells can reduce glycogen by cross-correction in the spinal cord, while little glycogen reduction is observed for native hGAA.
Conclusion
Taken together, the data in this example indicate that the engineered gene therapy constructs have significantly better tissue uptake and glycogen reduction, including effects in the brain and spinal cord, than wild-type GAA used in conventional therapy.
Example 8: animal study protocol
AAVhu68 Vector was generated and titrated by Penn Vector Core as described. (Lock, Alvira et al, 2010, "Rapid, simple, and university influencing of recombinant adono-associated viral vectors at scale," Hum Gene Ther 21(10): 1259-.
Gaa knockout mice (pompe mice) were purchased at Jackson Labs (stock #004154, also known as 6neo mice) in a C57BL/6/129 background creator.
Will receive 5X 10 in 0.1mL via the lateral tail vein11GC (about 2.5X 10)13GC/kg) mice of aavhu68.cag. hGAA (containing either native hGAA (SEQ ID NO:45) or engineered hGAA (SEQ ID NO:38)) were bled for serum separation on days 7 and 21 post vector administration and final bleeds (for plasma separation) were performed 28 days post injection and euthanized by bleeds. Tissue was collected rapidly, starting from the brain.
GAA Activity
Plasma was mixed with 5.6mM 4-MU- α -glucopyranoside pH 4.0 and incubated at 37 ℃ for three hours. The reaction was stopped with 0.4M sodium carbonate pH 11.5. Relative fluorescence units RFU were measured using a Victor3 fluorometer (355nm excitation and 460nm emission). Activity in nmol/mL/hr was calculated by interpolation from a 4-MU standard curve. The activity in individual tissue samples was further normalized based on the total protein content in the homogenate.
GAA signature peptides according to LC/MS
Plasma was precipitated in 100% methanol and centrifuged. The supernatant was discarded. The pellet was spiked with the unique stable isotope labeled peptide of hGAA as an internal standard and resuspended with trypsin and incubated for one hour at 37 ℃. Digestion was stopped with 10% formic acid. Tryptic peptides were separated by C-18 reverse phase chromatography and identified and quantified by ESI-mass spectrometry. The total GAA concentration in plasma was calculated from the tag peptide concentration.
Cell surface receptor binding assays
96-well plates were coated with receptor, washed and blocked with BSA. 28-day plasma from AAV-treated mice was serially diluted to give a series of reduced concentrations and incubated with the coupled receptor. After incubation, the plates were washed to remove any unbound hGAA and 4-MU- α -glucopyranoside was added at 37 ℃ for one hour. The reaction was stopped with 1.0M glycine pH 10.5 and the RFU read by a Spectramax fluorometer (excitation 370, emission 460). RFU was converted to activity (nmol/mL/hr) for each sample by interpolation according to a standard curve of 4-MU. Nonlinear regression was performed using GraphPad Prism.
Histology
Tissues were formalin fixed and paraffin embedded. Muscle sections were stained with PAS; CNS sections were stained with luxol fast blue/periodic acid-Schiff (PAS). Board certified veterinary pathologists (JH) blindly reviewed histological sections. Semi-quantitative estimates of the total percentage of cells with glycogen storage and cytoplasmic vacuolization were performed on the scanned sections. Scores from 0 to 4 are listed as described in the table below.
Immuno-histochemistry (IHC)
We studied transgene expression and cellular localization from sections immunostained with anti-human GAA antibody (Sigma HPA 029126).
Example 9: histology-tissue processing protocol and results in Pompe disease animal model
All tissues were fixed in 10% NBF (neutral buffered formalin). Assays (PAS and IHC) are routinely used in the art.
PAS staining of quadriceps and triceps (FIGS. 29 and 32)Tissues were fixed in 10% NBF and embedded in paraffin. Sections were post-fixed in 1% periodic acid and stained with Schiff reagent. Thereafter, the sections were counterstained with hematoxylin. Glycogen appears as magenta aggregates (lysosome binding) or diffuse pink (cytoplasm); the core is blue. From the images and assuming that each image represents a group, the ranking order with respect to glycogen clearance is: engineered hGAA>Natural hGAA. Throughout the image, the engineered hGAA construct produced more staining than the rest, indicating improved endocytosis of the GAA protein mediated by binding of vIGF2 to CI-MPR.
PAS staining of spinal cord (FIG. 26)Tissues were fixed in 10% NBF. Post-fixation in 1% periodic acid can be performed before or after paraffin embedding. Sections were stained with Schiff reagent and possibly counterstained with methylene blue. Glycogen appears as magenta aggregates (lysosome binding); the nerve fibers appear blue. The images focus on glycogen accumulation in the ventral horn of the spinal cord and motor neurons. In the constructs, the engineered hGAA appeared to be most effective in glycogen reduction.
GAAIHC (FIG. 22, FIG. 25, FIG. 27, FIG. 30 and FIG. 35)Tissues were fixed in 10% NBF and embedded in paraffin. Sections were incubated with anti-GAA primary antibody followed by a secondary antibody that recognized the primary antibody and carried enzyme-labeled HRP. Subsequently, an enzymatic reaction proceeds and a brown precipitated product is formed. The sections were then counterstained with hematoxylin. The construct showed GAA uptake into muscle fibers (fig. 31). Engineered hGAA>Natural hGAA. Throughout the image, the BiP-vIGF2 construct had more diffuse staining than the rest.
Engineered hGAA produced more GAA IHC signal than other vectors, with a spot-like appearance inside the muscle fibers, indicating far more efficient lysosomal targeting (fig. 22).
In summary, engineered hGAA consistently demonstrated superiority in tissue uptake, lysosomal targeting, and glycogen reduction in various tissues in the constructs.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Claims (151)
1. A gene therapy vector comprising a nucleic acid construct comprising:
(a) a nucleic acid sequence encoding a therapeutic protein, and
(b) a nucleic acid sequence encoding a peptide that binds with high affinity to cation-independent mannose 6-phosphate (M6P) receptor (CI-MPR).
2. The gene therapy vector of claim 1, wherein the peptide is a variant IGF2(vIGF2) peptide.
3. The gene therapy vector of claim 2, wherein the vIGF2 peptide comprises an amino acid sequence having at least 90% identity to SEQ ID No. 1 and having at least one substitution at one or more positions selected from the group consisting of: positions 6, 26, 27, 43, 48, 49, 50, 54, 55 and 65 of SEQ ID NO. 1.
4. The gene therapy vector of claim 3, wherein the at least one substitution is selected from the group consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R, L55R and K65R of SEQ ID NO. 1.
5. The gene therapy vector of claims 3-4, wherein the vIGF2 peptide comprises at least two substitutions at two or more positions selected from the group consisting of: positions 26, 27, 43, 48, 49, 50, 54 and 55 of SEQ ID NO. 1.
6. The gene therapy vector of claim 5, wherein the at least two substitutions are selected from the group consisting of: E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R, L55R and K65R of SEQ ID NO. 1.
7. The gene therapy vector of any one of claims 1-6, wherein the vIGF2 peptide comprises an N-terminal deletion at position 1 of SEQ ID No. 1.
8. The gene therapy vector of claim 7, wherein the vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID No. 1.
9. The gene therapy vector of any one of claims 1-8, wherein the vIGF2 peptide has reduced or no affinity for insulin receptor and IGFR1 compared to native IGF2 peptide.
10. The gene therapy vector of claim 9 and wherein the vIGF2 peptide is capable of promoting uptake of the therapeutic protein into lysosomes in cells.
11. The gene therapy vector of any one of claims 1-10, wherein the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder.
12. The gene therapy vector of claim 11, wherein the genetic disorder is a lysosomal storage disorder.
13. The gene therapy vector of claim 11 or claim 12, wherein the genetic disorder is selected from the group consisting of: aspartyl glucosamine uraemia, Barten disease (Batten disease), cystinosis, Fabry disease (Fabry disease), Gaucher disease type I (Gaucher disease), Gaucher disease type II, Gaucher disease type III, Pompe disease (Pompe disease), Tay Sachs disease (Tay Sachs disease), Sandhoff disease (Sandhoff disease), metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Leehler disease (Hurler disease), Hunter disease (Hunter disease), Sanphyspo disease type A (filappo disease), Sanphyr disease type B, Sanphyrperi disease type C, Pherpheri disease type D, Morquio disease type A (Morquio disease), Morphe disease type B, and Marmy-Nippon disease (Marmy disease), Mary-Nippon disease type A (Mary disease), Mary-type B (Mary-disease, Mary-Nippon disease, Mary-type A, Mary-type I, Mary disease, Mary-type I, and Mary type I, and Mar, Niemann-pick disease type B, niemann-pick disease type C1, niemann-pick disease type C2, sindler disease type I (Schindler disease), sindler disease type II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), Chronic Granulomatosis (CGD), CDKL5 deficiency, and neuronal ceroid lipofuscinosis.
14. The gene therapy vector of claim 11 or claim 12, wherein the genetic disorder is pompe disease.
15. The gene therapy vector of claim 11 or claim 12, wherein the genetic disorder is neuronal ceroid lipofuscinosis.
16. The gene therapy vector of any one of claims 1-15, wherein the therapeutic protein comprises an enzyme selected from the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof.
17. The gene therapy vector of claim 16, wherein the therapeutic protein is an alpha-glucosidase or an enzymatically active fragment thereof.
18. The gene therapy vector of claim 16, wherein the therapeutic protein is a palmitoyl protein thioesterase.
19. The gene therapy vector of claim 1, wherein the nucleic acid construct further comprises a translation initiation sequence.
20. The gene therapy vector of claim 19, wherein the translation initiation sequence comprises a Kozak sequence.
21. The gene therapy vector of claim 20, wherein the Kozak sequence comprises the sequence AX1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of which is any nucleotide.
22. The gene therapy vector of claim 21, wherein X1Contains A.
23. The gene therapy vector of claim 21 or claim 22, wherein X2Contains G.
24. The gene therapy vector of claim 21 or claim 22, wherein the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to AAGATGA (SEQ ID NO: 29).
25. The gene therapy vector of any one of claims 20-24, wherein the Kozak sequence comprises AAGATGA (SEQ ID NO: 29).
26. The gene therapy vector of claim 20, wherein the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to GCAAGATG (SEQ ID NO: 44).
27. The gene therapy vector of claim 20 or claim 26, wherein the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to GCAAGATG (SEQ ID NO: 44).
28. The gene therapy vector of claim 1, wherein the nucleic acid construct further comprises a signal nucleic acid sequence encoding a signal peptide, wherein the signal peptide is capable of increasing secretion of the therapeutic protein compared to the therapeutic protein without the signal peptide.
29. The gene therapy vector of claim 28, wherein the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide.
30. The gene therapy vector of claim 29, wherein the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17.
31. The gene therapy vector of claim 29, wherein the BiP signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 13-17.
32. The gene therapy vector of claim 28, wherein the signal peptide comprises a Gaussia signal peptide.
33. The gene therapy vector of claim 32, wherein the Gaussia signal peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32.
34. The gene therapy vector of claim 32, wherein the Gaussia signal peptide comprises SEQ ID NO: 32.
35. The gene therapy vector of any one of claims 1-34, wherein the vIGF2 nucleic acid sequence is 5' to the nucleic acid sequence encoding a therapeutic protein.
36. The gene therapy vector of any one of claims 1-34, wherein the vIGF2 nucleic acid sequence is 3' to the nucleic acid sequence encoding a therapeutic protein.
37. The gene therapy vector of any one of claims 1-36, wherein the nucleic acid construct further comprises a linker sequence encoding a linker peptide between the vIGF2 nucleotide sequence and the nucleic acid sequence encoding a therapeutic protein.
38. The gene therapy vector of claim 37, wherein the linker peptide comprises SEQ ID NOs 18-21, 33, or 37.
39. The gene therapy vector of any one of claims 1-38, wherein the gene therapy vector is a viral vector.
40. The gene therapy vector of claim 39, wherein the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, a poxviral vector, a vaccinia viral vector, an adenoviral vector, or a herpes viral vector.
41. A pharmaceutical composition comprising a therapeutically effective amount of the gene therapy vector of any one of claims 1 to 40 and a pharmaceutically acceptable carrier or excipient.
42. The pharmaceutical composition of claim 41, wherein the excipient comprises a non-ionic, low-permeability compound, buffer, polymer, salt, or combination thereof.
43. A method for treating a genetic disorder, the method comprising administering to a subject in need thereof a gene therapy vector of any one of claims 1 to 40 or a pharmaceutical composition of claim 41 or claim 42.
44. The method of claim 43, wherein the genetic disorder is a lysosomal storage disorder.
45. The method of claim 43 or claim 44, wherein the genetic disorder is selected from the group consisting of: aspartylglucosaminuria, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Muldoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hewler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Moquini disease type A, Moquine disease type B, Maruo-Lami disease, Spanish disease, Niemann-pick disease type A, Niemann-pick disease type B, Niemann-pick disease type C1, Niemann-pick disease type C2, Singler disease type I, Ocimum disease type II, Addison disease (CGA-severe combined immunodeficiency), and Addison disease (ADD), CDKL5 deficiency and neuronal ceroid lipofuscinosis.
46. The method of claim 43 or claim 44, wherein the genetic disorder is Pompe disease.
47. The method of claim 43 or claim 44, wherein the genetic disorder is neuronal ceroid lipofuscinosis.
48. The method of any one of claims 43-47, wherein the administering is performed intrathecally, intraocularly, intravitreally, transretinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraparenchymally, subcutaneously, or a combination thereof.
49. The method of any one of claims 43-47, wherein the administering is performed intrathecally.
50. A pharmaceutical composition comprising the gene therapy vector of any one of claims 1 to 40 and a pharmaceutically acceptable carrier or excipient for use in the treatment of a genetic disorder.
51. A pharmaceutical composition comprising the gene therapy vector of any one of claims 1 to 40 and a pharmaceutically acceptable carrier or excipient for use in the preparation of a medicament for the treatment of a genetic disorder.
52. The pharmaceutical composition of claim 50 or claim 51, wherein the genetic disorder is a lysosomal storage disorder.
53. The pharmaceutical composition of any one of claims 50-52, wherein the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hewlett-packard disease, Hunter disease, sanfilippo disease type A, sanfilippo disease type B, sanfilippo disease type C, sanfilippo disease type D, moquinase type A, moquinase type B, maratoto-lamic disease, sjgren disease, niemann-pick disease type A, niemann-pick disease type B, niemann-pick disease type C1, niemann-pick disease type C2, sinderler disease type I, sinderler disease type II, adenosine deaminase severe combined immunodeficiency disease (ADA-SCID), Chronic Granulomatosis (CGD), and neuronal ceroid lipofuscinosis.
54. The pharmaceutical composition of any one of claims 50-53, wherein the genetic disorder is Pompe disease.
55. The pharmaceutical composition of any one of claims 50-53, wherein the genetic disorder is neuronal ceroid lipofuscinosis.
56. The pharmaceutical composition of any one of claims 50-55, wherein the composition is formulated for intrathecal, intraocular, intravitreal, transretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, or subcutaneous administration.
57. The pharmaceutical composition of any one of claims 50-56, wherein the composition is formulated for intrathecal administration.
58. A gene therapy vector comprising a nucleic acid construct comprising, in 5 'to 3' order:
(a) a translation initiation sequence, and
(b) a nucleic acid sequence encoding a therapeutic protein.
59. The gene therapy vector of claim 58, wherein the translation initiation sequence comprises a Kozak sequence.
60. The gene therapy vector of claim 58, wherein the Kozak sequence comprises the sequence AX1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of which is any nucleotide.
61. The gene therapy vector of claim 59, wherein X1Contains A.
62. The gene therapy vector of claim 59 or claim 60, wherein X2Contains G.
63. The gene therapy vector of any one of claims 58-61, wherein the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to AAGATGA (SEQ ID NO: 29).
64. The gene therapy vector of any one of claims 58-62, wherein the Kozak sequence comprises AAGATGA (SEQ ID NO: 29).
65. The gene therapy vector of claim 58, wherein the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to GCAAGATG (SEQ ID NO: 44).
66. The gene therapy vector of claim 58 or claim 64, wherein the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to GCAAGATG (SEQ ID NO: 44).
67. The gene therapy vector of any one of claims 58-65, wherein the nucleic acid construct further comprises a nucleic acid sequence encoding a signal peptide capable of increasing secretion of the therapeutic protein compared to the therapeutic protein without the signal peptide.
68. The gene therapy vector of claim 66, wherein the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide.
69. The gene therapy vector of claim 67, wherein the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17.
70. The gene therapy vector of claim 67, wherein the BiP signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 13-17.
71. The gene therapy vector of claim 67, wherein the Gaussia signal peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32.
72. The gene therapy vector of claim 67, wherein the Gaussia signal peptide comprises the amino acid sequence of SEQ ID NO. 32.
73. The gene therapy vector of any one of claims 58-71, wherein the nucleic acid construct further comprises an Internal Ribosome Entry Sequence (IRES).
74. The gene therapy vector of claim 72, wherein the IRES is a cricket paralysis virus (CrPV) IRES.
75. The gene therapy vector of claim 72 or claim 73, wherein the IRES comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO 12.
76. The gene therapy vector of claim 72 or claim 73, wherein the IRES comprises SEQ ID NO 12.
77. A gene therapy vector comprising a nucleic acid construct comprising, in 5 'to 3' order:
(a) a nucleic acid sequence encoding a signal peptide, and
(b) a nucleic acid sequence encoding a therapeutic protein, wherein the signal peptide is capable of increasing secretion of the therapeutic protein compared to the therapeutic protein without the signal peptide.
78. The gene therapy vector of claim 76, wherein the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide.
79. The gene therapy vector of claim 77, wherein the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17.
80. The gene therapy vector of claim 77, wherein the BiP signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 13-17.
81. The gene therapy vector of any one of claims 76-79, wherein the signal peptide comprises a Gaussia signal peptide.
82. The gene therapy vector of claim 80, wherein the Gaussia signal peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32.
83. The gene therapy vector of claim 80, wherein the Gaussia signal peptide comprises SEQ ID NO 32.
84. The gene therapy vector of any one of claims 76-82, wherein the nucleic acid construct further comprises a translation initiation sequence.
85. The gene therapy vector of claim 83, wherein the translation initiation sequence comprises a Kozak sequence comprising sequence AX 1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of the above.
86. The gene therapy vector of claim 84, wherein X1Contains A.
87. The gene therapy vector of claim 84 or claim 85, wherein X2Contains G.
88. The gene therapy vector of any one of claims 84-86, wherein the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to AAGATGA (SEQ ID NO: 29).
89. The gene therapy vector of any one of claims 84-87, wherein the Kozak sequence comprises AAGATGA (SEQ ID NO: 29).
90. The gene therapy vector of claim 83, wherein the translation initiation sequence comprises a nucleic acid sequence having at least 90% identity to GCAAGATG (SEQ ID NO: 44).
91. The gene therapy vector of claim 83 or claim 89, wherein the translation initiation sequence comprises a nucleic acid sequence having at least 90% identity to GCAAGATG (SEQ ID NO: 44).
92. The gene therapy vector of any one of claims 76-90, wherein the nucleic acid construct further comprises an Internal Ribosome Entry Sequence (IRES).
93. The gene therapy vector of claim 91, wherein the IRES comprises an IRES selected from the group consisting of: cricket paralysis virus (CrPV) IRES, picornavirus IRES, foot and mouth disease virus IRES, Kaposi's sarcoma-associated herpesvirus IRES, hepatitis a IRES, hepatitis c IRES, pestivirus IRES, cricket paralysis virus (criptavirus) IRES, Rhopalosiphum palustris (Rhopalosiphum padi) virus IRES, and Merek's disease virus IRES.
94. The gene therapy vector of claim 91 or claim 92, wherein the IRES comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO 12.
95. The gene therapy vector of any one of claims 91-93, wherein the IRES comprises SEQ ID NO 12.
96. A gene therapy vector comprising a nucleic acid construct comprising, in 5 'to 3' order:
(a) an Internal Ribosome Entry Sequence (IRES), and
(b) a nucleic acid sequence encoding a therapeutic protein.
97. The gene therapy vector of claim 95, wherein the IRES comprises an IRES selected from the group consisting of: cricket paralysis virus (CrPV) IRES, picornavirus IRES, foot and mouth disease virus IRES, Kaposi sarcoma-associated herpesvirus IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, cricket paralysis virus IRES, gloomycosis graminearum virus IRES, and Merrex virus IRES.
98. The gene therapy vector of claim 96, wherein the IRES is a cricket paralysis virus (CrPV) IRES.
99. The gene therapy vector of claim 96 or claim 97, wherein the IRES comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO 12.
100. The gene therapy vector of any one of claims 96-98, wherein the IRES comprises SEQ ID NO 12.
101. The gene therapy vector of any one of claims 95-99, wherein the nucleic acid construct further comprises a translation initiation sequence.
102. The gene therapy vector of claim 100, wherein the translation initiation sequence comprises a Kozak sequence comprising AX1X2ATGA (SEQ ID NO:28), wherein X1And X2Each of which is any nucleotide.
103. The gene therapy vector of claim 101, wherein X1Contains A.
104. The gene therapy vector of claim 101 or claim 102, wherein X2Contains G.
105. The gene therapy vector of any one of claims 101-103, wherein the Kozak sequence comprises a nucleic acid sequence having at least 90% identity to AAGATGA (SEQ ID NO: 29).
106. The gene therapy vector of any one of claims 101-104, wherein the Kozak sequence comprises AAGATGA (SEQ ID NO: 29).
107. The gene therapy vector of claim 100, wherein the translation initiation sequence comprises a nucleic acid sequence having at least 90% identity to GCAAGATG (SEQ ID NO: 44).
108. The gene therapy vector of claim 100 or claim 106, wherein the translation initiation sequence comprises a nucleic acid sequence having at least 90% identity to GCAAGATG (SEQ ID NO: 44).
109. The gene therapy vector of any one of claims 95-107, wherein the nucleic acid construct further comprises a nucleic acid sequence encoding a signal peptide capable of increasing secretion of the therapeutic protein compared to the therapeutic protein without the signal peptide.
110. The gene therapy vector of claim 106, wherein the signal peptide is selected from the group consisting of a Binding Immunoglobulin Protein (BiP) signal peptide and a Gaussia signal peptide.
111. The gene therapy vector of claim 109, wherein the BiP signal peptide comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17.
112. The gene therapy vector of claim 109, wherein the BiP signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 13-17.
113. The gene therapy vector of claim 109, wherein the Gaussia signal peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 32.
114. The gene therapy vector of claim 109, wherein the Gaussia signal peptide comprises SEQ ID NO: 32.
115. The gene therapy vector of any one of claims 58-113, wherein the nucleic acid construct further comprises a nucleic acid sequence encoding an IGF2 peptide.
116. The gene therapy vector of claim 114, wherein the IGF2 peptide is a vIGF2 peptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: 1-11, 30-31, 34 and 35.
117. The gene therapy vector of claim 114, wherein the IGF2 peptide is a vIGF2 peptide comprising an amino acid sequence selected from the group consisting of: 1-11, 30-31, 34 and 35.
118. The gene therapy vector of any one of claims 114-116, wherein the vIGF2 nucleic acid sequence is 5' to a nucleic acid sequence encoding a therapeutic protein.
119. The gene therapy vector of any one of claims 114-116, wherein the vIGF2 nucleic acid sequence is 3' to a nucleic acid sequence encoding a therapeutic protein.
120. The gene therapy vector of any one of claims 114-118, wherein the nucleic acid construct further comprises a linker sequence encoding a linker peptide between the vIGF2 nucleotide sequence and the nucleic acid sequence encoding a therapeutic protein.
121. The gene therapy vector of claim 119, wherein the linker peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NOs 18-21, 33, or 37.
122. The gene therapy vector of claim 119, wherein the linker peptide comprises SEQ ID NOs 18-21, 33, or 37.
123. The gene therapy vector of any one of claims 58-121, wherein the therapeutic protein is selected from the group consisting of: alpha-galactosidase (A or B), beta-galactosidase, beta-hexosidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA alpha-glucosaminyl N-acetyltransferase, glucosaminoglycan alpha-L-iduronate hydrolase, heparan N-sulfatase, N-acetyl-alpha-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, and the like, Glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β -glucuronidase, hyaluronidase, α -N-acetylneuraminidase (sialidase), gangliosidase, phosphotransferase, α -glucosidase, α -D-mannosidase, β -D-mannosidase, aspartylglucosaminidase, α -L-fucosidase, batttenin, palmitoyl protein thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuron protein 6), or enzymatically active fragments thereof.
124. The gene therapy vector of claim 122, wherein the therapeutic protein is an alpha-galactosidase or enzymatically active fragment thereof.
125. The gene therapy vector of claim 122, wherein the enzyme is a palmitoyl protein thioesterase.
126. The gene therapy vector of claim 122, wherein the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder.
127. The gene therapy vector of claim 125, wherein the genetic disorder is a lysosomal storage disorder.
128. The gene therapy vector of claim 125, wherein the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease type A, san Francisella disease type B, san Francisella disease type C, san Francisella disease type D, morqui disease type A, morqui disease type B, Maruo-lamide disease, Spirosis, Niemann-pick disease type A, Niemann-pick disease type B, Niemann-pick disease type C1, Niemann-pick disease type C2, Sindler disease type I, Sindler disease type II, adenosine deaminase severe combined immunodeficiency (ADA-SCID), and neuronal ceroid lipofuscinosis.
129. The gene therapy vector of claim 125, wherein the genetic disorder is pompe disease.
130. The gene therapy vector of claim 125, wherein the genetic disorder is neuronal ceroid lipofuscinosis.
131. The gene therapy vector of any one of claims 58-129, wherein the gene therapy vector is a viral vector.
132. The gene therapy vector of claim 130, wherein the viral vector is an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a poxviral vector, a vaccinia viral vector, an adenoviral vector, or a herpesvirus vector.
133. A pharmaceutical composition comprising (i) a therapeutically effective amount of the gene therapy vector of any one of claims 58-131 and (ii) a pharmaceutically acceptable carrier or excipient.
134. The pharmaceutical composition of claim 132, wherein said carrier or excipient comprises a non-ionic low-permeability compound, buffer, polymer, salt, or combination thereof.
135. A method for treating a genetic disorder, the method comprising administering to a subject in need thereof a gene therapy vector of any one of claims 58-131 or a pharmaceutical composition of claim 132 or claim 133.
136. The method of claim 134, wherein the genetic disorder is a lysosomal storage disorder.
137. The method of claim 134 or claim 135, wherein the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease type A, san Francisella disease type B, san Francisella disease type C, san Francisella disease type D, morqui disease type A, morqui disease type B, Maruo-lamide disease, Spirosis, Niemann-pick disease type A, Niemann-pick disease type B, Niemann-pick disease type C1, Niemann-pick disease type C2, Sindler disease type I, Sindler disease type II, adenosine deaminase severe combined immunodeficiency (ADA-SCID), and neuronal ceroid lipofuscinosis.
138. The method of claim 134 or claim 135, wherein the genetic disorder is pompe disease.
139. The method of claim 134 or claim 135, wherein the genetic disorder is neuronal ceroid lipofuscinosis.
140. The method of any one of claims 134-138, wherein the administering is performed intrathecally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebroventricularly, intraparenchymally, ocularly, subcutaneously, or a combination thereof.
141. The method of any one of claims 134-139, wherein the administering is performed intrathecally.
142. A pharmaceutical composition comprising the gene therapy vector of any one of claims 58-131 and a pharmaceutically acceptable carrier or excipient for use in the treatment of a genetic disorder.
143. A pharmaceutical composition comprising the gene therapy vector of any one of claims 58-131 and a pharmaceutically acceptable carrier or excipient for use in the preparation of a medicament for the treatment of a genetic disorder.
144. The pharmaceutical composition of claim 141 or claim 142, wherein the genetic disorder is a lysosomal storage disorder.
145. The pharmaceutical composition of any one of claims 141-143, wherein the genetic disorder is selected from the group consisting of: aspartylglucamine uremia, Barten disease, cystinosis, Fabry disease, gaucher disease type I, gaucher disease type II, gaucher disease type III, Pompe disease, Tay-saxophone disease, Sandohoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Heller disease, hunter's disease, san Francisella disease type A, san Francisella disease type B, san Francisella disease type C, san Francisella disease type D, morqui disease type A, morqui disease type B, Maruo-lamide disease, Spirosis, Niemann-pick disease type A, Niemann-pick disease type B, Niemann-pick disease type C1, Niemann-pick disease type C2, Sindler disease type I, Sindler disease type II, adenosine deaminase severe combined immunodeficiency (ADA-SCID), and neuronal ceroid lipofuscinosis.
146. The pharmaceutical composition of any one of claims 141-144, wherein the genetic disorder is pompe disease.
147. The pharmaceutical composition of any one of claims 141-144, wherein the genetic disorder is neuronal ceroid lipofuscinosis.
148. The pharmaceutical composition of any one of claims 141-146, wherein said composition is formulated for intrathecal, intravenous, intramuscular, intraventricular, intracerebral, intracardial, ocular, or subcutaneous administration.
149. The pharmaceutical composition of any one of claims 141-147, wherein the composition is formulated for intrathecal administration.
150. A gene therapy vector comprising a nucleic acid construct comprising:
(a) a nucleic acid sequence encoding a therapeutic protein, and
(b) a nucleic acid sequence encoding a peptide that increases endocytosis of the therapeutic protein.
151. The gene therapy vector of claim 149, wherein the peptide that increases endocytosis of the therapeutic protein is a peptide that binds CI-MPR.
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